Patent Publication Number: US-2010129861-A1

Title: Isolation and Use of Novel Mammalian DExH Box Helicases

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to provisional application 61/112,832, filed Nov. 10, 2008, which is herein incorporated by reference in its entirety 
    
    
     FUNDING STATEMENT 
     The invention was made in the course of research supported by NIH grants, under Sponsored Assigned Identification Numbers 5R01GM059660 and 5R01AI051340. As a result, the U.S. government may have certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the isolation, purification, and use of novel mammalian DExH box helicases. In particular, the present invention relates to the isolation, purification and use of DHX29, a novel mammalian NTPase and RNA helicase. 
     BACKGROUND OF THE INVENTION 
     Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which typically requires at least 7 initiation factors (referred to as “eIFs”). These eIFs include eIFs 3, 2, 1, 1A, 4F, 4A and 4B, which cooperatively assist in formation of mRNAs. 
     Proteins, such as β-globin, serum albumin, myosin MYH6, and lysozyme, are encoded by mRNAs that have short, unstructured 5′ untranslated regions (5′-UTRs). These proteins are typically referred to as “house-keeping” proteins. In contrast, mRNAs that encode regulatory proteins such as proto-oncogenes, growth factors, their receptors, homeodomain proteins and transcription factors commonly have much longer 5′-UTRs that contain significant secondary structure. These proteins control many necessary processes, ranging from growth and development to innate immunity, cell cycle control, tumor invasion, and metastasis. Several translation initiation factors are over-expressed in tumors, which may cause cancer and/or affect its prognosis. 
     Current studies have focused on inhibitors of components of the eIF4F complex, and of pathways that signal to it as potential therapeutic targets for the treatment of cancers in mammals, particularly in humans. It had been determined that introduction of single GC-rich stems of increasing stability in a synthetic 5′ leader linked to a reporter open reading frame (ORF) progressively impaired translation of the reporter. However, it has now been discovered that the 7 eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5′-UTRs that are translated in mammalian cells. Moreover, 48S complexes assembled in vitro on β-globin mRNA using these 7 eIFs and analyzed by primer extension inhibition (“toe-printing”) have revealed incorrect fixation of mRNA on the A-site side of the mRNA-binding channel. Sufficient and efficient formation of the 48S complex is desirable. 
     There is currently a need for a method of isolating and using a novel mammalian helicase in various applications, including in initiating translation on various mRNAs (including 48S complex formation), which serves an important role in normal and abnormal cellular and developmental processes. Further, there is a need to prepare and isolate a purified form of the novel mammalian helicase, which may be useful in a variety of therapeutic applications. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the invention provides a method of forming a 48S complex comprising the use of a DExH-box protein. The DExH-box protein may be any protein desired, and may be DHX29. 
     In another embodiment of the invention, there is provided a method of purifying a DExH-box protein comprising the steps of: performing a ribosomal salt wash; precipitating a first fraction of the ribosomal salt wash containing the DExH-box protein; applying the first fraction to a DEAE column to provide an eluted fraction; performing a step elution on a plurality of aliquots of the eluted fraction through a phosphocellulose column to provide a step-eluted fraction; subjecting a step-eluted fraction to a first liquid chromatography column to provide a first purified fraction; subjecting the first purified fraction to a second liquid chromatography column to provide a second purified fraction; and applying the second purified fraction to a hydroxyapatite column to elute a purified DExH-box protein. 
     In yet another embodiment of the invention there is provided a method of performing translation initiation involving ribosomal scanning comprising the use of one particular helicase: DHX29. Other embodiments of the invention include providing biochemical assays of DHX29&#39;s activities that permit a means of identifying and assaying inhibitors of DHX29 function, providing a method of using DHX29 to achieve therapeutic regulation of gene expression and providing a method of using DHX29 as a biomarker for diagnosis of human cancer. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is one particular protocol for the purification of native DHX29. 
         FIG. 2  is a model of the domain organization of human DHX29. 
         FIG. 3  is an alignment of conserved motifs in the helicase core domains of human DHX29 and representative DExH-box proteins. 
         FIG. 4  is a characterization of purified native DHX29 (right lane) and protein molecular weight markers resolved by SDS-PAGE (left lane). 
         FIG. 5A  is a toe-printing analysis of 48S ribosomal initiation complexes assembled on β-globin mRNA. 
         FIG. 5B  is a toe-printing analysis of 48S ribosomal initiation complexes assembled on CAA-GUS and CAA (Stem)-GUS mRNAs. 
         FIG. 5C  is a toe-printing analysis of 48S ribosomal initiation complexes assembled on neutrophil cytosolic factor 2 mRNA (NCF2). 
         FIG. 5D  is a toe-printing analysis of 48S ribosomal initiation complexes assembled on Ser/Thr protein phosphatase CDC25 mRNA. 
         FIG. 5E  is an analysis of formation of elongation complexes on CAA-Stem-3,4-MVHC-STOP mRNAs assayed by toeprinting (left panel) and by sucrose density gradient (“SDG”) centrifugation with subsequent monitoring of  35 S-MVHC tetrapeptide (right panel). 
         FIG. 6A  is a toe-printing analysis of 48S complex assembly on β-globin mRNA. 
         FIG. 6B  is a toe-printing analysis of 48S complex assembly on mRNA containing two AUG triplets. 
         FIG. 6C  is a toe-printing analysis of 48S complex assembly on CAA-GUS Stem-1 mRNA. 
         FIG. 7A  is a depiction of association of purified DHX29 with individual 40S and 60S subunits, 80S ribosomes, 40S/eIF3/(CUUU) 9  complexes and 43S complexes containing 40S subunits and eIFs 2/3/1/1A. 
         FIG. 7B  is a depiction of association of purified DHX29 with yeast 40S subunits. 
         FIG. 7C  is a depiction of association of purified DHX29 with 40S/eIF3/(CUUU) 9  complexes in the presence/absence of nucleotides as indicated (lanes 4-7). 
         FIG. 7D  is a depiction of association of a DHX29 preparation containing a C-terminally truncated fragment resolved by SDS-PAGE (left panel) and its association with 40S subunits (right panel). 
         FIG. 8A  is a thin-layer chromatography analysis of DHX29&#39;s NTPase activity in the presence/absence of SDG-purified 43S complexes containing 40S subunits and eIF2/3/1/1A. 
         FIG. 8B  represents time courses of ATP hydrolysis by DHX29 in the presence/absence of (CUUU) 9  RNA, 18S rRNA, 43S complexes or 43S/(CUUU) 9 . 
         FIG. 8C  is a toe-printing analysis of 48S complexes assembled on CAA-GUS Stem-1 mRNA in the presence of SDG-purified 43S complexes, DHX29 and NTPs or non-hydrolyzable NTP analogues. 
         FIG. 9A  is a representation of non-denaturing PAGE done to show unwinding of 13-bp RNA duplexes with 25 nt-long single-stranded overhanging 5′-regions by DHX29, 43S complexes, 43S/DHX29 complexes and eIF4A/eIF4F. 
         FIG. 9B  is a representation of non-denaturing PAGE done to show unwinding of RNA duplexes corresponding to Stem-2, Stem-3 and Stem-4 with 25 nt-long single-stranded overhanging 5′-regions by DHX29, 43S complexes, 43S/DHX29 complexes and eIF4A/eIF4F. 
         FIG. 10A  is representation of SDG-purified 43S complexes containing different amounts of DHX29 and analyzed by SDS-PAGE and fluorescent SYPRO staining. 
         FIG. 10B  is a toe-printing analysis of 48S complex formation on CAA-GUS Stem-1 mRNA in the presence of SDG-purified free 43S complexes and different amounts of DHX29. 
         FIG. 10C  is a toe-printing analysis of 48S complex formation on CAA-GUS Stem-1 mRNA in the presence of DHX29-free 43S complexes, DHX29-saturated 43S complexes or DHX29-saturated 43S complexes and either DHX29-free 43S complexes or 43S/eIF3/(CUUU) 9  complexes. 
         FIG. 11A  is a toe-printing analysis of 40S/IRES binary complexes assembled on the CrPV IGR IRES. 
         FIG. 11B  is a toe-printing analysis of 40S/IRES binary complexes assembled on the CrPV IGR IRES. 
         FIG. 11C  is a toe-printing analysis of wt and Δdomain II CSFV IRESs. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention relates to the isolation, purification, and use of novel mammalian helicases, and in particular of DExH-box helicases (including DEAH-box helicases). In one embodiment, the invention relates to the isolation, purification, and use of one particular helicase, DHX29 [Seq. ID No. 1]. It has been determined that DHX29 is a novel putative initiation factor. It has been discovered that DHX29 may bind 40S subunits, which include the amino acid sequences forming ribosomal protein rpSA [Seq. ID No. 2], ribosomal protein rpS2 [Seq. ID No. 3], ribosomal protein rpS3 [Seq. ID No. 4], ribosomal protein rpS3a [Seq. ID No. 5], ribosomal protein rpS4X [Seq. ID No. 6], ribosomal protein rpS5 [Seq. ID No. 7], ribosomal protein rpS6 [Seq. ID No. 8], ribosomal protein rpS7 [Seq. ID No. 9], ribosomal protein rpS8 [Seq. ID No. 10], ribosomal protein rpS9 [Seq. ID No. 11], ribosomal protein rpS10 [Seq. ID No. 12], ribosomal protein rpS11 [Seq. ID No. 13], ribosomal protein rpS12 [Seq. ID No. 14], ribosomal protein rpS13 [Seq. ID No. 15], ribosomal protein rpS14 [Seq. ID No. 16], ribosomal protein rpS15 [Seq. ID No. 17], ribosomal protein rpS15A [Seq. ID No. 18], ribosomal protein rpS16 [Seq. ID No. 19], ribosomal protein rpS17 [Seq. ID No. 20], ribosomal protein rpS18 [Seq. ID No. 21], ribosomal protein rpS19 [Seq. ID No. 22], ribosomal protein rpS20 [Seq. ID No. 23], ribosomal protein rpS21 [Seq. ID No. 24], ribosomal protein rpS23 [Seq. ID No. 25], ribosomal protein rpS24 [Seq. ID No. 26], ribosomal protein rpS25 [Seq. ID No. 27], ribosomal protein rpS26 [Seq. ID No. 28], ribosomal protein rpS27 [Seq. ID No. 29], ribosomal protein rpS27A [Seq. ID No. 30], ribosomal protein rpS28 [Seq. ID No. 31], ribosomal protein rpS29 [Seq. ID No. 32], ribosomal protein rpS30 [Seq. ID No. 33], and the DNA sequence forming  H. sapiens  18S [Seq. ID No. 34]. Further, it has been discovered that DHX29 may hydrolyze ATP, GTP, UTP and CTP. Further, NTP hydrolysis by DHX29 has been found to be strongly stimulated by 43S complexes, and is required for DHX29&#39;s activity in promoting formation of the 48S complex. 
     Although the studies described herein relate to DHX29, it will be understood that the uses and isolation/purification methods described herein relate generally to DExH-box helicases generally, and are not limited to DHX29. DHX29 may be used in various functions, including aiding in forming a 48S initiation complex, following binding of a 43S preinitiation complex to the 5′-proximal region of a mRNA, in aiding in ribosomal scanning, and in ensuring fixation of mRNA in the ribosomal mRNA-binding cleft. As will be described in more detail below, DHX29 may be used alone or in combination with other complexes and/or eukaryotic initiation factors (eIFs). 
     As discussed herein, DExH-box proteins have been discovered to be useful in various therapeutic and beneficial applications. Although the DExH-box protein in any form may be useful, it is especially preferred to utilize the DExH-box protein in its purified form, to provide the most desirable and reproducible results. As used herein, the term “purified” refers to the protein in an apparently homogenous form, that is, at least about 95% pure, and more desirably at least about 98% pure. 
     Purification and/or Isolation of DExH-Box Proteins 
     The invention includes a protocol for isolating and purifying DExH-box proteins (such as DHX29) from mammalian cells. The protocol set forth herein may be used to isolate and purify DExH-box proteins from any mammalian cells, including human HeLa cells and rabbit reticulocytes. It will be understood that rabbit factors/ribosomal subunits may be interchangeably used for human equivalents, since the sequences are similar. Thus, the present invention may be directed to human initiation factors and their rabbit equivalents. The protocol set forth herein provides purified DExH-box proteins to a level of near-homogeneity, i.e., at least about 98% pure. 
     In one embodiment, the invention relates to a method of isolating and/or purifying a DExH-box protein, including DHX29. One preferred method 10 of purifying and isolating DHX29 is depicted in  FIG. 1 . In a first step  12 , a ribosomal salt wash may be prepared. Any desired ribosomal salt wash may be used, and in a preferred embodiment, the ribosomal salt wash may be prepared as described in Pisarev et al., Methods Enzymol., 430: 147-177 (2007), the contents of which are incorporated herein by reference. In this preparation, a polysomal suspension derived from a mammalian reticulocyte lysate may be stirred in a cooled environment, such as on ice. Although a ribosomal salt wash is preferred, it is contemplated that other starting materials may be used to purify DExH-box proteins involved in splicing, chromatin remodeling and other nuclear functions. 
     Thereafter, a desired amount of salt, preferably about 4 M, is added, preferably in a drop-wise manner. It may be desired to add a quantity of salt while continuously stirring the mixture. Any salt may be used, and in one embodiment, the salt is potassium chloride. The mixture is stirred continuously until there is approximately a 0.5 M salt final concentration. After further stirring, the suspension may be centrifuged. Desirably, the centrifuging is conducted in a Beckman Ti 50.2 rotor at approximately 45,000 rpm, for about 4.5 hours at 4° C., but any centrifugation technique desired may be used. The supernatant will be the ribosomal salt wash (“RSW”). 
     In a next step  14 , the fraction containing the DExH-box protein (such as DHX29) is then precipitated from the RSW, desirably with ammonium sulfate. DHX29 has been found to be in the 0-40% ammonium sulfate fraction, which may be prepared by adding ammonium sulfate (preferably in a powdered state) to the RSW while stirring the RSW. Desirably, the RSW is kept in a chilled state, and may be maintained on ice. Any amount of ammonium sulfate may be used, and desirably is added in an amount of about 240 g/L RSW. The resulting suspension may then be centrifuged. Centrifugation may be performed by any desired means, and preferably is conducted in a Sorvall SS34 rotor at approximately 15,000 rpm for about 20 minutes at about 4° C. The resulting product may then be removed from the apparatus. In one form, the resulting product is in pellet form, but may be in any resulting shape or state. The resulting product may then be dissolved in a 5-7 ml buffer (“buffer A”) with about 100 mM KCl. Preferably, the dissolved resulting product is dialyzed against 1 L of the buffer A overnight in a chilled state (at about 4° C.), and clarified by centrifugation at about 10,000 rpm for about 10 min at about 4° C. Any desired buffer A may be used, and desirably, the buffer A includes about 20 mM tris-HCl, having a pH of about 7.5, 2 mM DTT, 0.1 mM EDTA, and about 10% glycerol. 
     In a next step  16 , the dialyzed 0-40% ammonium sulfate fraction is then applied to a DEAE (DE52) column, equilibrated with buffer A and 100 mM salt. Preferably, the salt is KCl, but any desired salt may be used. The fraction containing DHX29 is then eluted in the flow-through fraction with buffer A and 100 mM salt. 
     In one embodiment, in a next step  18 , a plurality of aliquots, each of from 15-20 ml, of the resulting solution may then be applied to a phosphocellulose (P11) column. The aliquots are desirably equilibrated with buffer A and 100 mM salt. Step elution is then performed. In one embodiment, the step elution process may begin with buffer A and about 200 mM salt, followed by buffer A and about 300 mM salt; buffer A and about 400 mM salt; buffer A and about 500 mM salt; and buffer A and about 1000 mM salt. Any desired step elution may be performed, generally with increasing amounts of salt. Further, any number of aliquots may be used, preferably between 4-6 aliquots being used. 
     In a next step  20 , one fraction is selected and is then dialyzed overnight. In a desired embodiment, the fraction including buffer A and about 400 mM salt is dialyzed overnight, but any fraction may be used if desired. The dialyzation is preferably performed in a cooled environment, such as at about 4° C., against about 1 liter of a second buffer (“buffer B”) and 100 mM salt. Any desired salt may be used, and preferably the salt for this step  20  is the same as the salt used in previous steps. Buffer B may be the same or may be different than buffer A, and include any desired buffering material. Most desirably, buffer B differs from buffer A, and includes about 20 mM HEPES, with a pH of 7.5, 0.1 mM EDTA, 2 mM DTT, and 5% glycerol. After the overnight dialyzation, the fraction may then be loaded onto a liquid chromatography column. Preferably, the fraction is loaded onto a FPLC monoS HR 5/5 column that has previously been pre-equilibrated with buffer B and 100 mM salt. The target proteins to be purified may then be eluted with a mixture of buffer B and about 100-500 mM salt gradient. For embodiments where the target protein to be purified is DHX29, the preferable elution of DHX29 is at about 300 mM KCl (corresponding to fraction 28). 
     In the next step  22 , the eluted fraction from step  20  is then dialyzed overnight at about 4° C. It may be desired to dialyze the eluted fraction along with the neighboring fractions (generally corresponding to fractions 27 and 29). The fraction(s) are dialyzed against 1 liter of a third buffer (“buffer C”) and 100 mM salt. Buffer C may be the same or may be different from buffer A and/or buffer B, and may include any buffering mixture desired. Preferably, buffer C is different and includes about 20 mM Tris-HCl, with a pH of 7.5, 0.1 mM EDTA, 2 mM DTT, and 5% glycerol. After dialysis, the fraction may then be diluted with buffer C to 30 mM salt and loaded onto a liquid chromatography column. Desirably, the fraction is loaded onto a FPLC MonoQ HR 5/5 column, which has been pre-equilibrated with buffer C and 30 mM salt. The target protein may then be eluted with a mixture of buffer C and about 30-500 mM salt gradient. For embodiments where the target protein to be purified is DHX29, the preferable elution of DHX29 is at about 250 mM salt (which generally corresponds to fraction 27). 
     Finally, in the last step  24 , the eluted fraction of target protein is then dialyzed overnight in a cooled environment (such as at about 4° C.). It may be desired to dialyze the neighboring eluted fractions concurrently (generally corresponding to fractions 26 and 28). The eluted fraction(s) may be dialyzed against 1 liter of another buffer (“buffer D”). Buffer D may be the same or may be different than buffer A, buffer B, and/or buffer C, and may include any desired buffering mixture. Desirably, buffer D includes a mixture of about 20 mM Tris-HCl, with a pH of 7.5, 5% glycerol, and 100 mM salt. The dialyzed fraction may then be diluted approximately five-fold, with a 20 mM phosphate buffer. Any phosphate buffer may be used if desired, and desirably the phosphate buffer is a mixture of KH 2 PO 4  and K 2 HPO 4 , adjusting the pH to about 7.5 and adding about 5% glycerol. The sample may then be applied to a hydroxyapatite column, which is preferably pre-equilibrated with the phosphate buffer. The target proteins are then eluted with a 20-500 mM phosphate buffer gradient. For embodiments where the target protein to be purified is DHX29, the preferable elution of DHX29 is at about a 300 mM phosphate buffer (which generally corresponds to fraction 36). 
     The eluted product is then a substantially fully purified protein, and desirably is a substantially fully purified DExH-box protein, such as DHX29. 
     The process  10  set forth above provides one method of isolating and purifying target proteins, such as DExH-box proteins, including DHX29. Any process for purification and isolation of DHX29 may be incorporated if desired. For example, any or all of the above purification steps may be used if desired. For example, a substantially purified protein may be prepared without the last step  24  of exposure to a hydroxyapatite column. In other embodiments, one or more of steps  12 ,  14 ,  16 ,  18 ,  20 ,  22 , or  24  may be omitted if desired. It will be understood that, for optimal purification, each step should be performed, but any may be omitted if desired. In other embodiments, DHX29 can be over-expressed in  E. coli , yeast, insect cells or mammalian cells in recombinant form, with or without N-terminal or C-terminal affinity tags, which may be but are not limited to His6 (hexahistidine tag); GST (glutathione S-transferase); MBP (maltose-binding protein); FLAG (FLAG-tag peptide); BAP (biotin acceptor peptide); STREP (streptavidin-binding peptide); or CBP (calmodulin-binding peptide). Purification of recombinant DHX29 may include one or more of steps  12 ,  14 ,  16 ,  18 ,  20 ,  22 , and  24 , and will preferably include each of the listed steps. Further, purification of recombinant DHX29 may include the use of one or more appropriate affinity matrix, particularly if the recombinant DHX29 has N-terminal and/or C-terminal affinity tags. Use of such affinity matrix may aid in reducing the number of additional downstream steps to be used to fully purify the protein. 
     In addition, alternative steps may be performed if desired. For example, the process may include a gel-filtration step performed at any desired point in the process. Further, any desired ion-exchange columns and matrices may be used in place of or in combination with the Mono Q and Mono S columns described herein. Finally, alternative or additional buffer solutions may be used in the purification process outlined above. For example, buffer A may include mM tris in an amount of from 10-30 mM, having a pH of from about 6-9, having about 1-3 mM DTT, about 0.01-0.5 mM EDTA, and about 5-20% glycerol. As explained above, buffer B preferably differs from buffer A, and in one embodiment includes about 10-30 mM HEPES, with a pH of from 6-9, about 0.01-0.5 mM EDTA, 1-3 mM DTT, and 1-10% glycerol. Buffer C may be the same or may be different from any other buffer used, and may include about 10-30 mM Tris-HCl, with a pH of from 6-9, about 0.01-0.5 mM EDTA, 1-3 mM DTT, and 1-10% glycerol. Finally, buffer D may likewise be the same or may be different from any of the buffers used herein, and may include a mixture of about 10-30 mM Tris-HCl, with a pH of 6-9, 1-10% glycerol, and 50-150 mM salt. As will be understood, any of the buffers (A-D) may include any combination of the above components as desired. 
     The process described herein is not intended to be limited to the particular concentrations and compositions, and it is understood that equivalent columns, solutions, and equipment may be used if desired. 
     Structure of DHX29 
     DHX29 is a DExH-box mammalian RNA helicase. DHX29 is a 1369 amino-acid long, 155 kDa protein (Genbank accession NP — 061903) [Seq. ID No. 1]. DHX29 belongs to the DEx/HD box family of helicases, and particularly the DEAH subfamily of helicases. As depicted in  FIG. 2 , DHX29 contains a helicase domain  32 , a helicase associated domain of unknown function  34 , and an associated DUF1605 domain of unknown function  36 . The helicase domain (referenced as DExH) contains all of the consensus sequence motifs that are characteristic of DEAH helicases. DHX29 has C-terminally located helicase associated HA2 domain. A model of the conserved motifs of the helicase core domain of human DHX29 is set forth in  FIG. 3 . 
     The characterization of purified native DHX29 is depicted in  FIG. 4 . Various biochemical properties of DHX29 allow it to play various important roles in the initiation of translation (i.e., protein synthesis) in higher eukaryotes. In particular, DHX29 has ATPase, GTPase, CTPase and UTPase activities. In particular, the NTPase activity of DHX29 is weakly stimulated by random RNA, but is strongly stimulated by ribosomal 40S subunits, as set forth above, and by 18S ribosomal RNA. It has been determined that fully purified DHX29, such as that prepared by the process  10  described above, does not have processive helicase activity in the presence of any NTP, and further fully purified DHX29 binds to ribosomal 40S subunits in the absence of other translational components. Fully purified DHX29 is a stable constituent of ribosomal 43S complexes. 
     Table 1 below identifies DHX29 by LC/nanospray tandem mass-spectrometry of tryptic peptides. The amino acid residues are numbered according to the sequence of  H. sapiens  DHX29. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Identification of DHX29 
               
            
           
           
               
               
               
            
               
                   
                 Deduced Sequence 
                 Amino Acid Residues 
               
               
                   
                   
               
               
                   
                 SLEEEEKFDPNER 
                 251-263 [Seq. ID No. 35] 
               
               
                   
                   
               
               
                   
                 SPNPSFEK 
                 394-401 [Seq. ID No. 36] 
               
               
                   
                   
               
               
                   
                 DLFIAK 
                 489-494 [Seq. ID No. 37] 
               
               
                   
                   
               
               
                   
                 VVVVAGETGSGK 
                 590-601 [Seq. ID No. 38] 
               
               
                   
                   
               
               
                   
                 ASQTLSFQEIALLK 
                 1204-1217 [Seq. ID No. 39] 
               
               
                   
                   
               
               
                   
                 LACIVETAQGK 
                 1243-1253 [Seq. ID No. 40] 
               
               
                   
                   
               
               
                   
                 VLIDSVLR 
                 1334-1341 [Seq. ID No. 41] 
               
               
                   
                   
               
               
                   
                 ILQIITELIK 
                 1356-1365 [Seq. ID No. 42] 
               
               
                   
                   
               
            
           
         
       
     
     Table 2 below identifies the composition of ΔDHX29 by LC/nanospray tandem mass-spectrometry of tryptic peptides. The amino acid residues are numbered according to the sequence of  H. sapiens  DHX29 [Seq. ID No. 1]. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Identification of ΔDHX29 
               
            
           
           
               
               
               
            
               
                   
                 Deduced Sequence 
                 Amino Acid Residues 
               
               
                   
                   
               
               
                   
                 IIGVINEHK 
                  98-106 [Seq. ID No. 43] 
               
               
                   
                   
               
               
                   
                 SLEEEEKFDPNER 
                 251-263 [Seq. ID No. 44] 
               
               
                   
                   
               
               
                   
                 VVVVAGETGSGK 
                 590-601 [Seq. ID No. 45] 
               
               
                   
                   
               
               
                   
                 VCDELGCENGPGGR 
                 642-655 [Seq. ID No. 46] 
               
               
                   
                   
               
               
                   
                 NSLCGYQIR 
                 656-664 [Seq. ID No. 47] 
               
               
                   
                   
               
            
           
         
       
     
     Methods of Using DHX29 
     As will be described in more detail below, fully purified DHX29, such as that prepared by the purification process  10  described above, may promote proper fixation of mRNA in the mRNA-binding cleft of the ribosomal 40S subunit in 48S initiation complexes assembled at the initiation codon. Such proper fixation may be apparent in toe-printing analyses of 48S complexes assembled on native capped β-globin mRNA [Seq. ID No. 48] as suppression of aberrant toe-prints at positions +8-9 nt relative to the AUG initiation codon (A=+1) and enhancement of correct toe-prints at positions +15-17 nt that correspond to the leading edge of the 40S subunit. A toe-printing analysis of 48S ribosomal initiation complexes assembled on β-globin mRNA is depicted in  FIG. 5A . 
     Further, DHX29 further enhances the formation of 48S initiation complexes by several means. First, DHX29 may be used to enhance the process of ribosomal scanning, functioning synergistically with eIF4A [Seq. ID No. 49]/eIF4B [Seq. ID No. 50]/eIF4F (which is a heterotrimer comprising eIF4A [Seq. ID No. 49], eIF4E [Seq. ID No. 51] and eIF4G [Seq. ID No. 52]) to enhance scanning on synthetic and/or natural mRNAs with highly structured 5′-UTRs. In addition, DHX29 may be used to functionally replace any or all of eIF4A/eIF4B/eIF4F to promote scanning on mRNAs with 5′-UTRs with weak or no significant secondary structure in their 5′-UTRs. Such use may be important as scanning may not effectively occur without DHX29 on mRNAs with the most highly structured 5′-UTRs. In addition, DHX29 ensures correct fixation of mRNA in the ribosomal mRNA-binding channel of 48S complexes after scanning, following arrest at the initiation codon, thus increasing the proportion of correctly assembled 48S complexes. These and other features will be more adequately and thoroughly described in the Examples set forth below. 
     As will be described in more detail in the Examples, in conjunction with other defined components of the translation apparatus (including but not limited to 40S ribosomal subunits, initiator tRNA [Seq. ID No. 53], GTP, ATP and eukaryotic initiation factors such as eIF1 [Seq. ID No. 54], eIF1A [Seq. ID No. 55], eIF2 (which is comprised of three subunits: subunit 1 [Seq. ID No. 56], subunit 2 [Seq. ID No. 57], subunit 3 [Seq. ID No. 58]), eIF3 (which is comprised of thirteen subunits: eIF3A [Seq. ID No. 59], eIF3B [Seq. ID No. 60], eIF3C [Seq. ID No. 61], eIF3D [Seq. ID No. 62], eIF3E [Seq. ID No. 63], eIF3F [Seq. ID No. 64], eIF3G [Seq. ID No. 65], eIF3H [Seq. ID No. 66], eIF3I [Seq. ID No. 67], eIF3J [Seq. ID No. 68], eIF3K [Seq. ID No. 69], eIF3L [Seq. ID No. 70], eIF3M [Seq. ID No. 71]), eIF4A [Seq. ID No. 49], eIF4B [Seq. ID No. 50], eIF4E [Seq. ID No. 51], and eIF4G [Seq. ID No. 52]), fully purified DHX29 may enable ribosomal 43S preinitiation complexes assembled with the above components to scan synthetic mRNA 5′-UTRs that contain modest secondary structure. Toe-printing analysis of 48S ribosomal initiation complexes assembled on CAA-GUS mRNAs containing stems of various stabilities is depicted in  FIG. 5B . Further, fully purified DHX29 may enable ribosomal 43S preinitiation complexes assembled with the above components to scan the wild-type 5′-UTRs of natural mRNAs that contain extensive secondary structure, such as neutrophil cytosolic factor 2 mRNA (as shown in  FIG. 5C ) [Seq. ID No. 72] or CDC25 mRNA (as shown in  FIG. 5D ) [Seq. ID No. 73]. 
     DHX29 may be used to aid in various processes and mechanisms. In one embodiment, DHX29 may be used in the synthesis of eukaryotic proteins. Eukaryotic protein synthesis typically begins with the assembly of 48S initiation complexes at the initiation codon of mRNA, which typically requires at least seven initiation factors (eIFs). First, various initiation factors bind to the 40S subunit to form a 43S preinitiation complex. eIF4F, eIF4A and eIF4B cooperatively unwind the cap-proximal region of mRNA allowing attachment of the 43S complexes. The 43S preinitiation complex (comprising a 40S ribosomal subunit, initiator tRNA, eIF2, eIF3, eIF1 and eIF1A) then attaches to the 5′-proximal region of the unwound mRNA. Attachment of a 43S complex is typically mediated by eIF4F (which, as set forth above, comprises eIF4E, eIF4A and eIF4G), eIF4A and eIF4B. In addition, eIF4F, eIF4A and eIF4B also assist 43S complexes during scanning. 
     In typical processes, the ribosomal subunits then scan along the 5′-UTR to the initiation codon where they stop, forming 48S complexes with established P-site codon-anticodon base pairing. It will be understood that “scanning” refers to unwinding of secondary structure in the 5′ leader, 5′-3′ movement of the 43S complex, and monitoring of interactions between the tRNA Met   i  [Seq. ID No. 53] anticodon and triplets in the leader to prevent codon-anticodon mismatches and to signal establishment of correct base-pairing so that eIF2 hydrolyzes its bound GTP and loses affinity for Met-tRNA Met   i . 
     However, eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4B and eIF4F (collectively referred to as eIFs) have been found to be insufficient alone for an efficient and practical 48S complex formation on mRNAs with long structured 5′-UTRs. In particular, eIFs do not generally support high-level formation of 48S complexes on mRNAs containing longer and more stable stems, such as CAA-GUS stem-3 [Seq. ID No. 74] and stem-4 [Seq. ID No. 75] mRNAs ( FIG. 5B ). In addition, eIFs support only very weak 48S complex assembly on cellular neutrophil cytosolic factor 2 (NCF2) mRNA [Seq. ID No. 72] containing a 168 nt-long 5′-UTR. Finally, eIFs have been found not to be sufficient to promote 48S complex formation at all on Ser/Thr protein phosphatase CDC25 mRNA [Seq. ID No. 73], which contains a 271 nt-long 5′-UTR. 
     Due to the lack of sufficiency of eIFs on long, highly-structured mRNAs, DExH-box proteins, in particular DHX29, may be used in this process to more efficiently form a 48S complex on mRNAs with long structured 5′-UTRs. Inclusion of DHX29 in an in vitro reconstituted system has been found to strongly increase 48S complex formation on such mRNAs. Specifically, DHX29 may be used to bind 40S subunits and provide a stable constituent of 43S complexes. Further, DHX29 aids in forming the 48S complex by efficiently hydrolyzing ATP, GTP, UTP and CTP. Further, NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes. In this fashion, DHX29 may be used to greatly aid in the formation of 48S complexes. Use of DHX29 may be used to increase 48S complex formation by a significant amount. In some embodiments, the use of DHX29 may increase 48S formation by any amount from at least 2-fold up to about 40-fold. In a preferred embodiment, use of DHX29 increases 48S complex formation from about 3-fold to about 20-fold. DHX29 may be controlled to increase 48S formation to any amount desired, including at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold and at least 40-fold. 
     In one embodiment, DHX29 may be incorporated into the 48S complex forming process in addition to the traditional eIFs described above. Alternatively, DHX29 may be included in 48S complex formation in the absence of any or all of the eIFs, including but not limited to eIF4A, eIF4B and eIF4F. DHX29 and at least eIF4F, with or without eIF4B synergistically promote efficient 48S complex formation on mRNAs with structured and stable 5′-UTRs. In some processes, such as those including NCF2 or CDC25 mRNAs, DHX29 and at least eIF4F, with or without eIF4B, and may be used in conjunction to promote 48S complex formation. In some embodiments, DHX29 may be used to assist with ribosomal scanning, and not used during initial attachment of 43S complexes. In some embodiments, DHX29 may be used for efficient 48S complex formation on mRNAs with highly structured 5′-UTRs and also to suppress the aberrant +8-9 nt toe-print. 
     In other uses, DHX29 may be bound to ribosomal complexes so as to include conformational changes near the mRNA-binding cleft that accommodate the 3′-portion of mRNA. In some embodiments, DHX29 may be used to increase leaky scanning, thus enhancing 48S complex formation on the second AUG codon of mRNA containing two AUG triplets, irrespective of the presence of eIF1 or eIF1A. 
     The DExH-box protein DHX29 may be used as a factor that is required for efficient initiation on mRNAs with long structured 5′-UTRs, which typically encode regulatory proteins. DHX29 may additionally be used to modify altered ribosomal conformations to enhance the processivity of scanning complexes. Further, DHX29 may be used to stabilize binding of mRNA in the mRNA-binding channel of the 40S subunit near its entrance. Finally, DHX29 may be used to remodel ribonucleo-protein complexes without extensive unwinding of RNA duplexes. 
     In some embodiments, analysis of DHX29 levels may be used in diagnosing diseases, including but not limited to cancer. Further, inhibition of DHX29 itself may be used in treating such diseases. The requirement for initiation factors between different mRNAs is non-uniform. Further, translation of some mRNAs is dependent on the activity of factors that promote ribosomal attachment to and scanning on mRNA. Consequently, translation of some mRNAs may be selectively and disproportionately affected by inhibition of the activity of these factors or down-regulation of their expression levels. Prior research has established that mRNAs that encode proteins that are involved in different aspects of malignancy are particularly dependent on eIF4F. As such, agents that block the activities of eIF4F and its components may thus be used as potential therapeutic agents for such malignancy. 
     Translation of mRNAs with long, structured 5′UTRs (which includes mRNAs encoding proteins that promote cell growth, cell cycle progression, inhibition of cell death and tumor growth and innate immune responses) is dependent on DHX29. In contrast, translation of ‘house-keeping’ mRNAs is not dependent on DHX29. Further, translation of CDC25, a regulator of the cell cycle, has been found to be dependent on DHX29. Consequently, DHX29 may thus be used as a target for therapeutic intervention. 
     In one embodiment of the present invention, inhibitors of DHX29&#39;s biochemical activities (such as nucleotide binding and hydrolysis, binding to the ribosomal 40S subunit, promoting ribosomal scanning and correct assembly of 48S complexes on mRNA) may be used as therapeutic agents in the treatment of cancer. Assays of DHX29&#39;s biochemical activities that are required for its ability to mediate translation of mRNAs with long and highly structured 5′UTRs may be used to identify potential specific inhibitors of DHX29. Further, such assays may be used to test their inhibition of DHX29&#39;s activity in translation initiation. Through experimentation described in the Examples below, for example, it has been found that GMP-PNP and AMP-PNP, inhibitors of DHX29&#39;s NTPase activity, specifically block its function in translation initiation ( FIG. 8C , compare lanes 4, 6 with 8, 9). 
     Thus, DHX29 may be used as a biomarker for cancerous tissues. As with many eIFs, the number of molecules of DHX29 in cells is lower than the number of ribosomes. Thus, just as a reduction in levels of active DHX29 by inhibitors would inhibit translation of proteins that promote malignancy, enhanced levels of DHX29 may promote expression of such proteins. Moreover, DHX29 may be upregulated in malignant melanomas, lymphomas, ovarian endometroid carcinoma and ovarian serous adenocarcinoma. 
     Levels of DHX29 protein may be determined by various means, such as by western blot (as in  FIG. 7D ), which may then be compared against levels in control cells/tissues. Using such comparative data, levels of DHX29 mRNA may be determined and compared relative to standards using quantitative RT-PCR, which are conducted using primers designed on the basis of the human DHX29 sequence (Genbank NM — 019030). Other comparative means may be used as desired. 
     The methods and uses described herein may be more clearly understood from a consideration of the non-limiting Examples provided herein. 
     EXAMPLES 
     1. Efficient 48S Complex Formation on mRNAs with Structured 5′-UTRs with DHX29 
     Although eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4B, and eIF4F promote efficient 48S complex formation on model synthetic mRNAs comprising the β-glucuronidase (GUS) coding region and an unstructured 5′-UTR consisting of 19 CAA repeats (CAA-GUS mRNA [Seq. ID No. 76]), they did not support high level 48S complex formation on CAA-GUS Stem-3 and Stem 4 mRNAs containing more stable stems with ΔG=−18.9 and −27.6 kcal/mol, respectively. ( FIG. 5B , lanes 18 and 24). Further, these eIFs also supported only very weak 48S complex assembly on neutrophil cytosolic factor 2 (NCF2) mRNA containing a 168 nt-long 5′UTR ( FIG. 5C , lane 3). Finally, they did not promote 48S complex formation at all on CDC25 mRNA containing a 271 nt-long 5′-UTR ( FIG. 5D , lane 2). 
     Extensive purification from RRL of missing factor(s) required for efficient 48S complex formation on such structured 5′-UTRs was undertaken. Purification yielded an apparently homogeneous ˜150 kDa protein, as depicted in  FIG. 4 , which was identified as DHX29, a putative DExH-box helicase ( FIG. 2 ). 
     Experiments were conducted to determine the effect of DHX29 in an in vitro reconstituted system, which were found to increase 48S complex formation on both CAA-GUS Stem 3 and Stem-4 mRNAs ( FIG. 5B , lanes 19 and 25, respectively) and on NCF2 mRNA ( FIG. 5C , lane 4), and further allowed 48S complex formation on CDC25 mRNA ( FIG. 5D , lane 1). DHX29 also slightly (by about 20-30%) stimulated the already efficient 48S complex formation on CAA-GUS Stem-1 [Seq. ID No. 77] and Stem-2 [Seq. ID No. 78] mRNAs ( FIG. 5B , lanes 10, 15). It was discovered that moderate stimulation of 48S complex formation on Stem-containing CAA-GUS mRNAs by DHX29 occurred even in the absence of eIF4A, eIF4B, and eIF4F ( FIG. 5B , lanes 3, 8, 13, 17 and 23), but was lower than by eIF4A, eIF4B and eIF4F ( FIG. 5B , lanes 4, 9, 14, 18 and 24). DHX29 promoted only marginal 48S complex assembly on β-globin mRNA in the absence of eIF3F, eIF4B and eIF4A ( FIG. 5A , lanes 5, 6). 
     2. Verification that 48S Complexes Assembled with DHX29 are Elongation-Competent 
     Experiments were conducted to verify that 48S complexes assembled with DHX29 were elongation-competent. To this end, formation of ribosomal complexes was assayed on derivatives of CAA-GUS Stem-3 and Stem-4 mRNAs encoding a MVHC tetrapeptide followed by a UAA stop codon. Addition of 60S subunits, eIF5 and eIF5B, elongation factors and aminoacylated tRNAs to 48S complexes assembled on both mRNAs with DHX29 yielded prominent toe prints +16-17 nt from the UGC Cys codon that occupies the P-site of elongating ribosomes arrested at the stop codon. This can be seen in  FIG. 5E , left panel. As with 48S complexes, substantially more elongation complexes formed on both mRNAs in the presence of DHX29, assayed by toe-printing and sucrose density gradient centrifugation ( FIG. 5E , right panel). 
     3. 48S Complex Formation on β-Globin mRNA 
     It is known that eIF2, eIF3, eIF1, eIF1A, eIF4A, eIF4B and eIF4F ensure adequate 48S complex formation on native capped β-globin mRNA. Additional toe-prints appeared +8-9 nt downstream of the AUG codon, at least as much as 30-40% of the level of the +15-17 nt toe-prints corresponding to properly assembled 48S complexes ( FIG. 6A , lane 2). The +8-9 toe-prints were apparent on other mRNAs, for example on the first AUG codon of mRNA containing two AUG triplets [Seq. ID No. 79] surrounded by CAA repeats ( FIG. 6B , lanes 2, 4). In contrast, 48S complexes assembled on β-globin or other mRNAs in RRL yielded toe-prints exclusively at +15-17 positions ( FIG. 6A , lane 3). Appearance of the +8-9 toe-print required 40S subunits, Met-tRNA Met   i , eIFs and an AUG codon, thus suggesting that it corresponds to a 48S complex in which the 3′-portion of mRNA is not fixed in the 40S subunit&#39;s mRNA-binding cleft, thus allowing reverse transcriptase to penetrate further. In addition, formation of the +8-9 toe-print was also eIF1-dependent, and was exacerbated by some eIF1A mutants. Almost no such toe print was observed on the first AUG codon of mRNA with two AUG triplets in reaction mixtures lacking eIF1 ( FIG. 6B , compare lanes 2, 4 and 6, 8). 
     DHX29 was used for formation of 48S complex formation on β-globin mRNA. Although DHX29 was found not to be absolutely essential for 48S complex formation on β-globin mRNA, it was discovered that DHX29 used in 48S complex formation on β-globin mRNA allowed a more efficient 48S complex formation. It was found that DHX29 used in this capacity suppressed the aberrant +8-9 toe-print, and had the same effect upon delayed addition to preformed initiation complexes ( FIG. 5A , lanes 3, 4). Further, DHX29 also suppressed the aberrant +8-9 nt toe-print on other mRNAs, including the mRNA with two AUG triplets ( FIG. 6B , lanes 1, 3). 
     Thus, it was determined that binding of DHX29 to ribosomal complexes induces conformational changes near the mRNA-binding cleft that accommodate the 3′-portion of mRNA. DHX29 additionally increased leaky scanning, enhancing 48S complex formation on the second AUG codon of mRNA containing two AUG triplets, irrespective of the presence of eIF1 or eIF1A ( FIG. 6B , lanes 1, 3, 5, 7). In reaction mixtures lacking eIF4F, eIF4A, and eIF4B, DHX29 was found to promote low-level 48S complex formation on CAA-GUS Stem-1 even without eIF1 and eIF1A ( FIG. 6C , lane 3). However, eIF1, particularly in combination with eIF1A, substantially increased initiation ( FIG. 6C , lanes 5, 6). 
     4. Interactions Between DHX29 and Translational Components 
     Experiments were conducted to identify interactions between DHX29 and translational components that could drive DHX29 to ribosomal complexes. These experiments demonstrated that DHX29 is capable of binding stably to 40S subunits. DHX29 was also found not to bind to 60S or 80S ribosomes. Further, experiments showed that DHX29 remained associated with the 40S subunits during sucrose density gradient centrifugation ( FIG. 7A , lanes 4, 5, 7). DHX29 was found to associate with 40S subunit monomers, but not to the dimers that occur in mammalian 40S subunit preparations ( FIG. 7A , lanes 6, 7). 
     It was further discovered that DHX29 bound stably and stoichiometrically to 40S/eIF3 complexes, including those formed with (CUUU) 9  RNA. Further, DHX29 was found to bind stably to 43S complexes ( FIG. 7A , lanes 8, 9). Further, DHX29 was found to bind stably and stoichiometrically to yeast 40S subunits ( FIG. 7B ). Experiments revealed that DHX29&#39;s ribosomal binding is nucleotide-independent ( FIG. 7C ), and as much DHX29 associated with 40S/eIF3 complexes in the presence or absence of ATP, ADP, or AMPPNP. In RRL, DHX29 was present in 40S-containing ribosomal complexes. Further, truncated DHX29 was prepared, which are identified as containing a ˜90-95 kDa band ( FIG. 7D , left panel). 
     5. The Ribosomal Position of DHX29 
     To obtain insight into the ribosomal position of DHX29, experiments were conducted. Experiments revealed that the region of DHX29 responsible for ribosomal binding is located in the N-terminal two thirds of the protein. In particular, chemical and enzymatic foot-printing of 18S rRNA in 43S and 43S/DHX29 complexes were compared. It was found that DHX29 strongly protected CUC 527-9  and UUU 530-2  in the apical region of helix (h) 16 from RNase VI cleavage and CMCT modification, respectively. Further, DHX29 was found to weakly protect the neighboring A 526  from DMS modification. Finally, DHX29 did not protect G 534  on the opposite strand of the stem from RNase T1 cleavage. In eukaryotic 40S subunits, h16 is rotated towards the back of the 40S subunit, pointing into the solvent. If the observed protections resulted from direct interaction between h16 and DHX29, rather than from induced conformational changes, then DHX29 is found to bind to the 40S subunit near the mRNA entrance. 
     6. Characterization of DHX29 NTPase Activity 
     The NTPase activity of DHX29 was characterized to fully define the biochemical properties of DHX29. DHX29 was found to lack nucleotide specificity and hydrolyzed ATP, GTP, CTP, and UTP, which all lack the Q-motif upstream of the helicase domain that has been implicated in determining the specificity of adenine recognition by related DEAD box helicases ( FIG. 8A ). 
     DHX29&#39;s NTPase activity was strongly stimulated by 43S complexes, whereas stimulation by single-stranded RNA was low ( FIGS. 8A ,  8 B). 18S rRNA had higher stimulatory activity than (CUUU) 9  RNA, but lower than 43S complexes ( FIG. 8B ). The greatest level of stimulation occurred in the presence of 43S complexes with (CUUU) 9  RNA. 
     eIF4A/eIF4B/eIF4F-independent 48S complex assembly on CAA-GUS Stem-1 mRNA was then investigated in the presence of DHX29 and different NTPs ( FIG. 8C ). 43S complexes formed with eIF2/eIF3/eIF1/eIF1A were then separated from unincorporated GTP by sucrose density gradient centrifugation and incubated with DHX29 and mRNA in the presence and absence of GTP, ATP, CTP, UTP, GMPPNP or AMPPNP. It was found that the highest stimulation of DHX29 was with GTP or ATP, and was slightly lower with CTP or UTP ( FIG. 8C , lanes 4-7). It was determined that NTP hydrolysis by DHX29 may therefore be required for its activity in 48S complex formation. 
     7. Potential Helicase Activity of DHX29 
     Experiments were conducted to investigate the potential helicase activity of DHX29. RNA duplexes comprising overhanging 25 nt-long 5′ or 3′-ends and 13 nt-long or 10 nt-long double stranded regions (ΔG=−21 and −14.6 kCal/mol, respectively) as well as corresponding blunt duplexes and duplexes resembling stems 2, 3, and 4 of CAA-GUS Stem-2-4 mRNAs. It was found that DHX29 did not unwind 13 nt-long duplexes with overhanging 5′- or 3′-ends in the presence of NTP, whereas unwinding by eIF4A/eIF4F was efficient ( FIG. 9A , left panel). There was found weak unwinding of these duplexes by isolated 43S/DHX29 complexes ( FIG. 9A , right panel, lane 2). Additionally, there was found marginal unwinding (i.e., less than 5%) by DHX29 of 10 nt-long duplexes with overhanging ends. DHX29 was found to unwind Stem-2 duplex ( FIG. 9B , lane 3). Further, Stem-3 duplex unwinding by DHX29 was marginal ( FIG. 9B , lane 6). 
     8. DHX29 Participation in Multiple Rounds of 48S Complex Formation 
     DHX29 was found to stimulate 48S complex formation most strongly when it was present in substoichiometric amounts relative to 43S complexes. The most active in 48S complex assembly on GAA-GUS Stem-1 mRNA were sucrose density gradient-purified 43 S/DHX29 complexes having a ratio of 43S to DHX29 of about 10:1 ( FIG. 10A , lane 3). Complexes with 43S:DHX29 ratios of from about 2:1 to about 1:1 were found to be progressively less active ( FIG. 10A , lanes 4, 5). 
     A mixture of DHX29-free 43S complexes and 43S/DHX29-saturated 43S complexes that individually had low activities were found to together promote very efficient 48S complex formation ( FIG. 10B , lanes 4, 5). As such, a proportion of DHX29 may be inactive, but the DHX29 from active 43S/DHX29 complexes may have beneficial activities, including being able to dissociate from ribosomal complexes and participating in new rounds of initiation. 
     In an alternative, it was found that stimulation of 48S complex formation by DHX29 may require dissociation from the 40S subunit at a point in the process before the 48S complex is formed. In this embodiment, the excess of free 43S complexes would ensure rebinding of dissociated DHX29 to a new 43S complex. To investigate this embodiment, DHX29-saturated 43S complexes were mixed with purified complex of 40S, eIF3 and (CUUU) 9 . The complex of 40S, eIF3 and (CUUU) 9  were found to not stimulate 48S complex formation by 43S/DHX29 complexes ( FIG. 10C , lanes 3, 5). 
     9. Influence of DHX29 on 48S Complex Formation During IRES-Mediated Initiation 
     As set forth above, DHX29 may be used in aiding 48S complex formation during IRES-mediated initiation if this process involves internal ribosomal entry followed by scanning, but may impair initiation if this process involves direct binding of the ribosome to the initation codon, for example on the intergenic region (IGR) IRES of Dicstroviruses such as Cricket paralysis virus (CrPV) and the Heptatitis C virus (PCV)-like IRESs of vlaviviruses such as Classical swine fever virus (CSFV) and picornaviruses such as Simian Picornavirus type 9 (SPV9). Experiments were conducted to determine the influence of DHX29 on such 48S complex formation. Generally, binding of the CrPV IRES [Seq. ID No. 80] to 40S subunits yields two sets of toe-prints, one corresponding to the leading edge of the 40S subunit +15-16 nt from the P-site CCU codon (at AG 6228-9 ), and one corresponding to a second IRES-40S subunit interaction (at AA 6161-2 ). When present in stoichiometric amounts relative to 40S subunits, DHX29 was found to almost abrogate the toe-prints at AG 6228-9  irrespective of whether DHX29 was added before CrPV IRES mRNA ( FIG. 11A ) or to preassembled IRES/40S complexes ( FIG. 11B ). 
     Binding of CSFV IRES [Seq. ID No. 81] to 40S subunits also yields two sets of toe-prints, the first corresponding to the leading edge of the 40S subunit +15-17 nt from the P-site AUG codon (at UUU 387-9 ) and a second corresponding to a contact of the 40S subunit with the pseudoknot of the IRES (at C 334 ). Again, DHX29 was found to strongly reduce the toe-prints at UUU 387-9  in 40S/CSFV IRES complexes, irrespective of when it was added ( FIG. 11C ). DHX29 was found to have less effect on toe-prints corresponding to 40S/IRES contacts outside the mRNA-binding cleft than on toe-prints at the leading edge of the bound 40S subunit. 
     Even upon delayed addition, DHX29 was found to abrogate toe-prints corresponding to 48S complexes assembled on the CSFV IRES in the presence of eIF2, eIF3 and Met-tRNA Met   i  ( FIG. 11C ). Deletion of IRES domain II [Seq. ID No. 82] was found to eliminate the sensitivity of 48S complexes to dissociation by eIF1. Although deletion of domain II did not completely suppress the dissociating effect of DHX29, 48S complexes assembled on the IRES lacking domain II were less sensitive to DHX29 than complexes assembled on the wt IRES ( FIG. 11C , lanes 5-7 and 12-14). 48S complexes assembled on the HCV-like IRES of Simian picornavirus type 9, which are much more resistant to dissociation by eIF1, were resistant to dissociation by DHX29. 
     10. Purification of Native DHX29 
     DHX29 was purified from the 0-40% ammonium sulphate precipitation fraction of the 0.5M KCl ribosomal salt wash from 2 liters of rabbit reticulocyte lysate (RRL). The pellet was resuspended in buffer A (20 mM Tris-HCl, pH 7.5, 10% glycerol, 2 mM DTT, 0.1 mM EDTA) containing 100 mM KCl and applied to a DEAE (D52) column equilibrated with buffer A+100 mM KCl. The fraction containing DHX29 was eluted in the flow-through fraction with buffer A+100 mM KCl. This fraction was applied to a phosphocellulose (P11) column equilibrated with buffer A+100 mM KCl. Step elution was done with buffer A containing 100, 200, 300, 400 and 500 mM KCl. DHX29 eluted at 300-400 mM KCl. This fraction was dialyzed overnight against buffer B (20 mM HEPES, pH 7.5, 5% glycerol, 2 mM DTT, 0.1 mM EDTA) containing 100 mM KCl and then applied to a FPLC MonoS HR 5/5 column. Fractions were collected across a 100-500 mM KCl gradient. DHX29 eluted at ±300 mM KCl. DHX29-containing fractions were dialyzed overnight against buffer C (20 mM Tris-HCl, pH 7.5, 5% glycerol, 2 mM DTT, 0.1 mM EDTA) containing 100 mM KCl and then applied to a FPLC MonoQ HR 5/5 column. Fractions were collected across a 100-500 mM KCl gradient. DHX29 eluted at ˜250 mM KCl. DHX29-containing fractions were dialyzed overnight against buffer containing 20 mM Tris-HCl, pH 7.5, 5% glycerol and 100 mM KCl, then diluted 5-fold with 20 mM phosphate buffer, pH 7.5 with 5% glycerol and applied to a hydroxyapatite column pre-equilibrated in the same phosphate buffer. Fractions were collected across a 20-500 mM phosphate buffer gradient. Apparently homogenous DHX29 eluted at ˜300 mM phosphate buffer. The identity of DHX29 was confirmed by LC-nanospray tandem mass spectrometry of peptides derived by in-gel tryptic digestion at the Rockefeller University Proteomics Resource Center. 
     It should be understood that various alternatives to the embodiments of the present invention described herein can be employed in practicing the present invention. It is intended that the following claims define the scope of the present invention and that structures and methods within the scope of these claims and their equivalents be covered entirely. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Listings of Sequences in the Present Application 
               
            
           
           
               
               
               
               
            
               
                 Seq. ID 
                   
                 Description of 
                   
               
               
                 No. 
                 Deduced Sequence 
                 Sequence 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 mggknkkhka paaavvraav sasraksaea giageaqskk pvsrpataaa 
                 DHX29 
                   
               
               
                   
                 aaagsreprv kqgpkiysfn stndssgpan ldksilkvvi nnkleqriig 
               
               
                   
                 vinehkkqnn dkgmisgrlt akklqdlyma lqafsfktkd iedamtntll 
               
               
                   
                 yggdlhsald wlclnlsdda lpegfsqefe eqqpksrpkf qspqiqatis 
               
               
                   
                 pplqpktkty eedpkskpkk eeknmevnmk ewilryaeqq 
               
               
                   
                 neeeknensk sleeeekfdp nerylhlaak lldakeqaat fkleknkqgq 
               
               
                   
                 keaqekirkf qremetledh pvfnpamkis hqqnerkkpp vategesaln 
               
               
                   
                 fnlfeksaaa teeekdkkke phdvrnfdyt arswtgkspk qflidwvrkn 
               
               
                   
                 lpkspnpsfe kvpvgrywkc rvrviksedd vlvvcptilt edgmqaqhlg 
               
               
                   
                 atlalyrlvk gqsvhqllpp tyrdvwlews daekkreeln kmetnkprdl 
               
               
                   
                 fiakllnklk qqqqqqqqhs enkrensedp eeswenlvsd edfsalsles 
               
               
                   
                 anvedlepvr nlfrklqstp kyqkllkerq qlpvfkhrds ivetlkrhrv 
               
               
                   
                 vvvagetgsgkstqvphfll edlllnewea skcnivctqp rrisavslan 
               
               
                   
                 rvcdelgcen gpggrnslcg yqirmesrac estrllyctt gvllrklqed 
               
               
                   
                 gllsnvshvi vdevhersvq sdflliilke ilqkrsdlhl ilmsatvdse 
               
               
                   
                 kfstyfthcp ilrisgrsyp vevfhledii eetgfvlekd seycqkflee 
               
               
                   
                 eeevtinvts kaggikkyqe yipvqtgaha dlnpfyqkys srtqhailym 
               
               
                   
                 nphkinldli lellayldks pqfrniegav liflpglahi qqlydllsnd 
               
               
                   
                 rrfyserykv ialhsilstq dqaaaftlpp pgvrkivlat niaetgitip 
               
               
                   
                 dvvfvidtgr tkenkyhess qmsslvetfv skasalqrqg ragrvrdgfc 
               
               
                   
                 frmytrerfe gfmdysvpei lrvpleelcl himkcnlgsp edflskaldp 
               
               
                   
                 pqlqvisnam nllrkigace lnepkltplg qhlaalpvnv kigkmlifga 
               
               
                   
                 ifgcldpvat laavmteksp fttpigrkde adlaksalam adsdhltiyn 
               
               
                   
                 aylgwkkarq eggyrseity crrnflnrts lltledvkqe liklvkaagf 
               
               
                   
                 sssttstswe gnrasqtlsf qeiallkavl vaglydnvgk iiytksvdvt 
               
               
                   
                 eklaciveta qgkaqvhpss vnrdlqthgw llyqekirya rvylrettli 
               
               
                   
                 tpfpvllfgg dievqhrerl lsidgwiyfq apvkiavifk qlrvlidsvl 
               
               
                   
                 rkklenpkms lendkilqii teliktenn 
               
               
                   
               
               
                 2 
                 msgaldvlqm keedvlkfla agthlggtnl dfqmeqyiyk rksdgiyiin 
                 Ribosomal 
               
               
                   
                 lkrtweklll aaraivaien padvsvissr ntgqravlkf aaatgatpia 
                 Protein rpSA 
               
               
                   
                 grftpgtftn qiqaafrepr llvvtdprad hqplteasyv nlptialcnt 
               
               
                   
                 dsplryvdia ipcnnkgahs vglmwwmlar evlrmrgtis 
               
               
                   
                 rehpwevmpd lyfyrdpeei ekeeqaaaek avtkeefqge wtapapefta 
               
               
                   
                 tqpevadwse gvqvpsvpiq qfptedwsaq patedwsaap taqatewvga 
               
               
                   
                 ttdws 
               
               
                   
               
               
                 3 
                 maddagaagg pggpggpgmg nrggfrggfg sgirgrgrgr grgrgrgrga 
                 Ribosomal 
               
               
                   
                 rggkaedkew mpvtklgrlv kdmkikslee iylfslpike seiidfflga 
                 Protein rpS2 
               
               
                   
                 slkdevlkim pvqkqtragq rtrfkafvai gdynghvglg vkcskevata 
               
               
                   
                 irgaiilakl sivpvrrgyw gnkigkphtv pckvtgrcgs vlvrlipapr 
               
               
                   
                 gtgivsapvp kkllmmagid dcytsargct atlgnfakat fdaisktysy 
               
               
                   
                 ltpdlwketv ftkspyqeft dhlvkthtrv svqrtqapav att 
               
               
                   
               
               
                 4 
                 mavqiskkrk fvadgifkae lnefltrela edgysgvevr vtptrteiii 
                 Ribosomal 
               
               
                   
                 latrtqnvlg ekgrrirelt avvqkrfgfp egsvelyaek vatrglcaia 
                 protein rpS3 
               
               
                   
                 qaeslrykll gglavrracy gvlrfimesg akgcevvvsg klrgqraksm 
               
               
                   
                 kfvdglmihs gdpvnyyvdt avrhvllrqg vlgikvkiml pwdptgkigp 
               
               
                   
                 kkplpdhvsi vepkdeilpt tpiseqkggk peppampqpv pta 
               
               
                   
               
               
                 5 
                 mavgknkrlt kggkkgakkk vvdpfskkdw ydvkapamfn 
                 Ribosomal 
               
               
                   
                 irnigktlvt rtqgtkiasd glkgrvfevs ladlqndeva frkfklited 
                 protein rpS3a 
               
               
                   
                 vqgkncltnf hgmdltrdkm csmvkkwqtm ieahvdvktt dgyllrlfcv 
               
               
                   
                 gftkkrnnqi rktsyaqhqq vrqirkkmme imtrevqtnd lkevvnklip 
               
               
                   
                 dsigkdieka cqsiyplhdv fvrkvkmlkk pkfelgklme lhgegsssgk 
               
               
                   
                 atgdetgakv eradgyeppv qesv 
               
               
                   
               
               
                 6 
                 margpkkhlk rvaapkhwml dkltgvfapr pstgphklre clpliiflrn 
                 Ribosomal 
               
               
                   
                 rlkyaltgde vkkicmqrfi kidgkvrtdi typagfmdvi sidktgenfr 
                 protein rpS4X 
               
               
                   
                 liydtkgrfa vhritpeeak yklckvrkif vgtkgiphlv thdartiryp 
               
               
                   
                 dplikvndti qidletgkit dfikfdtgnl cmvtgganlg rigvitnrer 
               
               
                   
                 hpgsfdvvhv kdangnsfat rlsnifvigk gnkpwislpr gkgirltiae 
               
               
                   
                 erdkrlaakq ssg 
               
               
                   
               
               
                 7 
                 mtewetaapa vaetpdiklf gkwstddvqi ndislqdyia vkekyakylp 
                 Ribosomal 
               
               
                   
                 hsagryaakr frkaqcpive rltnsmmmhg rnngkklmtv rivkhafeii 
                 protein rpS5 
               
               
                   
                 hlltgenplq vlvnaiinsg predstrigr agtvrrqavd vsplrrvnqa 
               
               
                   
                 iwllctgare aafrniktia ecladelina akgssnsyai kkkdelerva ksnr 
               
               
                   
               
               
                 8 
                 mklnisfpat gcqklievdd erklrtfyek rmatevaada lgeewkgyvv 
                 Ribosomal 
               
               
                   
                 risggndkqg fpmkqgvlth grvrlllskg hscyrprrtg erkrksvrgc 
                 protein rpS6 
               
               
                   
                 ivdanlsvln lvivkkgekd ipgltdttvp rrlgpkrasr irklfnlske 
               
               
                   
                 ddvrqyvvrk plnkegkkpr tkapkiqrlv tprvlqhkrr rialkkqrtk 
               
               
                   
                 knkeeaaeya kllakrmkea kekrqeqiak rrrlsslras tsksessqk 
               
               
                   
               
               
                 9 
                 mfsssakivk pngekpdefe sgisqallel emnsdlkaql relnitaake 
                 Ribosomal 
               
               
                   
                 ievgggrkai iifvpvpqlk sfqkiqvrlv relekkfsgk hvvfiaqrri 
                 protein rpS7 
               
               
                   
                 lpkptrksrt knkqkrprsr tltavhdail edlvfpseiv gkrirvkldg 
               
               
                   
                 srlikvhldk aqqnnvehkv etfsgvykkl tgkdvnfefp efql 
               
               
                   
               
               
                 10 
                 mgisrdnwhk rrktggkrkp yhkkrkyelg rpaantkigp rrihtvrvrg 
                 Ribosomal 
               
               
                   
                 gnkkyralrl dvgnfswgse cctrktriid vvynasnnel vrtktlvknc 
                 protein rpS8 
               
               
                   
                 ivlidstpyr qwyeshyalp lgrkkgaklt peeeeilnkk rskkiqkkyd 
               
               
                   
                 erkknakiss lleeqfqqgk llaciasrpg qcgradgyvl egkelefylr 
               
               
                   
                 kikarkgk 
               
               
                   
               
               
                 11 
                 mpvarswvcr ktyvtprrpf eksrldqelk ligeyglrnk revwrvkftl 
                 Ribosomal 
               
               
                   
                 akirkaarel ltldekdprr lfegnallrr lvrigvldeg kmkldyilgl 
                 protein rpS9 
               
               
                   
                 kiedflerrl qtqvfklgla ksihharvli rqrhirvrkq vvnipsfivr 
               
               
                   
                 ldsqkhidfs lrspygggrp grvkrknakk gqggagagdd eeed 
               
               
                   
               
               
                 12 
                 mlmpkknria iyellfkegv mvakkdvhmp khpeladknv 
                 Ribosomal 
               
               
                   
                 pnlhvmkamq slksrgyvke qfawrhfywy ltnegiqylr dylhlppeiv 
                 protein rpS10 
               
               
                   
                 patlrrsrpe tgrprpkgle gerparltrg eadrdtyrrs avppgadkka 
               
               
                   
                 eagagsatef qfrggfgrgr gqppq 
               
               
                   
               
               
                 13 
                 madiqteray qkqptifqnk krvllgetgk eklpryykni glgfktpkea 
                 Ribosomal 
               
               
                   
                 iegtyidkkc pftgnvsirg rilsgvvtkm kmqrtivirr dylhyirkyn 
                 protein rpS11 
               
               
                   
                 rfekrhknms vhlspcfrdv qigdivtvge crplsktvrf nvlkvtkaag 
               
               
                   
                 tkkqfqkf 
               
               
                   
               
               
                 14 
                 maeegiaagg vmdvntalqe vlktalihdg largireaak aldkrqahlc 
                 Ribosomal 
               
               
                   
                 vlasncdepm 
                 protein rpS12 
               
               
                   
                 yvklvealca ehqinlikvd dnkklgewvg lckidregkp rkvvgcscvv 
               
               
                   
                 vkdygkesqa kdvieeyfkc kk 
               
               
                   
               
               
                 15 
                 mgrmhapgkg lsqsalpyrr svptwlklts ddvkeqiykl akkgltpsqi 
                 Ribosomal 
               
               
                   
                 gvilrdshgv aqvrfvtgnk ilrilkskgl apdlpedlyh likkavavrk 
                 protein rpS13 
               
               
                   
                 hlernrkdkd akfrlilies rihrlaryyk tkrvlppnwk yesstasalv a 
               
               
                   
               
               
                 16 
                 maprkgkekk eeqvislgpq vaegenvfgv chifasfndt fvhvtdlsgk 
                 Ribosomal 
               
               
                   
                 eticrvtggm kvkadrdess pyaamlaaqd vaqrckelgi talhiklrat 
                 protein rpS14 
               
               
                   
                 ggnrtktpgp gaqsalrala rsgmkigrie dvtpipsdst rrkggrrgrr l 
               
               
                   
               
               
                 17 
                 maeveqkkkr tfrkftyrgv dldqlldmsy eqlmqlysar qrrrlnrglr 
                 Ribosomal 
               
               
                   
                 rkqhsllkrl rkakkeappm ekpevvkthl rdmiilpemv gsmvgvyngk 
                 protein rpS15 
               
               
                   
                 tfnqveikpe mighylgefs itykpvkhgr pgigathssr fiplk 
               
               
                   
               
               
                 18 
                 mvrmnvlada lksinnaekr gkrqvlirpc skvivrfltv mmkhgyigef 
                 Ribosomal 
               
               
                   
                 eiiddhragk ivvnltgrln kcgvisprfd vqlkdlekwq nnllpsrqfg 
                 protein rpS15A 
               
               
                   
                 fivlttsagi mdheearrkh tggkilgfff 
               
               
                   
               
               
                 19 
                 mpskgplqsv qvfgrkktat avahckrgng likvngrple mieprtlqyk 
                 Ribosomal 
               
               
                   
                 llepvlllgk erfagvdirv rvkggghvaq iyairqsisk alvayyqkyv 
                 protein rpS16 
               
               
                   
                 deaskkeikd iliqydrtll vadprrcesk kfggpgarar ygksyr 
               
               
                   
               
               
                 20 
                 mgrvrtktvk kaarviieky ytrlgndfht nkrvceeiai ipskklrnki 
                 Ribosomal 
               
               
                   
                 agyvthlmkr iqrgpvrgis iklqeeerer rdnyvpevsa ldqeiievdp 
                 protein rpS17 
               
               
                   
                 dtkemlklld fgslsnlqvt qptvgmnfkt prgpv 
               
               
                   
               
               
                 21 
                 mslvipekfq hilrvlntni dgrrkiafai taikgvgrry ahvvlrkadi 
                 Ribosomal 
               
               
                   
                 dltkragelt edeverviti mqnprqykip dwflnrqkdv kdgkysqvla 
                 protein rpS18 
               
               
                   
                 ngldnklred lerlkkirah rglrhfwglr vrgqhtkttg rrgrtvgvsk kk 
               
               
                   
               
               
                 22 
                 mpgvtvkdvn qqefvralaa flkksgklkv pewvdtvkla khkelapyde 
                 Ribosomal 
               
               
                   
                 nwfytraast arhlylrgga gvgsmtkiyg grqrngvmps hfsrgsksva 
                 protein rpS19 
               
               
                   
                 rrvlqalegl kmvekdqdgg rkltpqgqrd ldriagqvaa ankkh 
               
               
                   
               
               
                 23 
                 mafkdtgktp vepevaihri ritltsrnvk slekvcadli rgakeknlkv 
                 Ribosomal 
               
               
                   
                 kgpvrmptkt lrittrktpc gegsktwdrf qmrihkrlid lhspseivkq 
                 protein rpS20 
               
               
                   
                 itsisiepgv evevtiada 
               
               
                   
               
               
                 24 
                 mqndagefvd lyvprkcsas nriigakdha siqmnvaevd kvtgrfngqf 
                 Ribosomal 
               
               
                   
                 ktyaicgair rmgesddsil rlakadgivs knf 
                 protein rpS21 
               
               
                   
               
               
                 25 
                 mgkcrglrta rklrshrrdq kwhdkqykka hlgtalkanp fggashakgi 
                 Ribosomal 
               
               
                   
                 vlekvgveak qpnsairkcv rvqlikngkk itafvpndgc lnfieendev 
                 protein rpS23 
               
               
                   
                 lvagfgrkgh avgdipgvrf kvvkvanvsl lalykgkker prs 
               
               
                   
               
               
                 26 
                 mndtvtirtr kfmtnrllqr kqmvidvlhp gkatvpktei reklakmykt 
                 Ribosomal 
               
               
                   
                 tpdvifvfgf rthfgggktt gfgmiydsld yakknepkhr larhglyekk 
                 protein rpS24 
               
               
                   
                 ktsrkqrker knrmkkvrgt akanvgagkk pke 
               
               
                   
               
               
                 27 
                 mppkddkkkk dagksakkdk dpvnksggka kkkkwskgkv 
                 Ribosomal 
               
               
                   
                 rdklnnlvlf dkatydklck evpnyklitp avvserlkir gslaraalqe 
                 protein rpS25 
               
               
                   
                 llskgliklv skhraqviyt rntkggdapa ageda 
               
               
                   
               
               
                 28 
                 mtkkrrnngr akkgrghvqp irctncarcv pkdkaikkfv irniveaaav 
                 Ribosomal 
               
               
                   
                 rdiseasvfd ayvlpklyvk lhycvscaih skvvrnrsre arkdrtpppr 
                 protein rpS26 
               
               
                   
                 frpagaaprp ppkpm 
               
               
                   
               
               
                 29 
                 mplakdllhp speeekrkhk kkrlvqspns yfmdvkcpgc ykittvfsha 
                 Ribosomal 
               
               
                   
                 qtvvlcvgcs tvlcqptggk arltegcsfr rkqh 
                 protein rpS27 
               
               
                   
               
               
                 30 
                 akkrkkksyt tpkknkhkrk kvklavlkyy kvdengkisr lrrecpsdec 
                 Ribosomal 
               
               
                   
                 gagvfmashf drhycgkccl tycfnkpedk 
                 protein rpS27A 
               
               
                   
               
               
                 31 
                 mdtsrvqpik larvtkvlgr tgsqgqctqv rvefmddtsr siirnvkgpv 
                 Ribosomal 
               
               
                   
                 regdvltlle serearrlr 
                 protein rpS28 
               
               
                   
               
               
                 32 
                 mghqqlywsh prkfgqgsrs crvcsnrhgl irkyglnmcr qcfrqyakdi 
                 Ribosomal 
               
               
                   
                 gfikld 
                 protein rpS29 
               
               
                   
               
               
                 33 
                 kvhgslarag kvrgqtpkva kqekkkkktg rakrrmqynr rfvnvvptfg 
                 Ribosomal 
               
               
                   
                 kkkgpnans 
                 protein rpS30 
               
               
                   
               
               
                 34 
                 tacctggttg atcctgccag tagcatatgc ttgtctcaaa gattaagcca 
                   H. sapiens  18S 
               
               
                   
                 tgcatgtctg agtacgcacg gccggtacag tgaaactgcg aatggctcat 
               
               
                   
                 taaatcagtt atggttcctt tggtcgctcg ctcctctcct acttggataa 
               
               
                   
                 ctgtggtaat tctagagcta atacatgccg acgggcgctg acccccttcg 
               
               
                   
                 cgggggggat gcgtgcattt atcagatcaa aaccaacccg gtcagcccct 
               
               
                   
                 ctccggcccc ggccgggggg cgggcgccgg cggctttggt gactctagat 
               
               
                   
                 aacctcgggc cgatcgcacg ccccccgtgg cggcgacgac ccattcgaac 
               
               
                   
                 gtctgcccta tcaactttcg atggtagtcg ccgtgcctac catggtgacc 
               
               
                   
                 acgggtgacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 
               
               
                   
                 ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga 
               
               
                   
                 cccggggagg tagtgacgaa aaataacaat acaggactct ttcgaggccc 
               
               
                   
                 tgtaattgga atgagtccac tttaaatcct ttaacgagga tccattggag 
               
               
                   
                 ggcaagtctg gtgccagcag ccgcggtaat tccagctcca atagcgtata 
               
               
                   
                 ttaaagttgc tgcagttaaa aagctcgtag ttggatcttg ggagcgggcg 
               
               
                   
                 ggcggtccgc cgcgaggcga gccaccgccc gtccccgccc cttgcctctc 
               
               
                   
                 ggcgccccct cgatgctctt agctgagtgt cccgcggggc ccgaagcgtt 
               
               
                   
                 tactttgaaa aaattagagt gttcaaagca ggcccgagcc gcctggatac 
               
               
                   
                 cgcagctagg aataatggaa taggaccgcg gttctatttt gttggttttc 
               
               
                   
                 ggaactgagg ccatgattaa gagggacggc cgggggcatt cgtattgcgc 
               
               
                   
                 cgctagaggt gaaattcttg gaccggcgca agacggacca gagcgaaagc 
               
               
                   
                 atttgccaag aatgttttca ttaatcaaga acgaaagtcg gaggttcgaa 
               
               
                   
                 gacgatcaga taccgtcgta gttccgacca taaacgatgc cgaccggcga 
               
               
                   
                 tgcggcggcg ttattcccat gacccgccgg gcagcttccg ggaaaccaaa 
               
               
                   
                 gtctttgggt tccgggggga gtatggttgc aaagctgaaa cttaaaggaa 
               
               
                   
                 ttgacggaag ggcaccacca ggagtggagc ctgcggctta atttgactca 
               
               
                   
                 acacgggaaa cctcacccgg cccggacacg gacaggattg acagattgat 
               
               
                   
                 agctctttct cgattccgtg ggtggtggtg catggccgtt cttagttggt 
               
               
                   
                 ggagcgattt gtctggttaa ttccgataac gaacgagact ctggcatgct 
               
               
                   
                 aactagttac gcgacccccg agcggtcggc gtcccccaac ttcttagagg 
               
               
                   
                 gacaagtggc gttcagccac ccgagattga gcaataacag gtctgtgatg 
               
               
                   
                 cccttagatg tccggggctg cacgcgcgct acactgactg gctcagcgtg 
               
               
                   
                 tgcctaccct acgccggcag gcgcgggtaa cccgttgaac cccattcgtg 
               
               
                   
                 atggggatcg gggattgcaa ttattcccca tgaacgagga attcccagta 
               
               
                   
                 agtgcgggtc ataagcttgc gttgattaag tccctgccct ttgtacacac 
               
               
                   
                 cgcccgtcgc tactaccgat tggatggttt agtgaggccc tcggatcggc 
               
               
                   
                 cccgccgggg tcggcccacg gccctggcgg agcgctgaga agacggtcga 
               
               
                   
                 acttgactat ctagaggaag taaaagtcgt aacaaggttt ccgtaggtga 
               
               
                   
                 acctgcggaa ggatcatta 
               
               
                   
               
               
                 35 
                 sleeeekfdpner 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (251-263) 
               
               
                   
               
               
                 36 
                 spnpsfek 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (394-401) 
               
               
                   
               
               
                 37 
                 dlfiak 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (489-494) 
               
               
                   
               
               
                 38 
                 vvvvagetgsgk 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (590-601) 
               
               
                   
               
               
                 39 
                 asqtlsfqeiallk 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (1204-1217) 
               
               
                   
               
               
                 40 
                 lacivetaqgk 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (1243-1253) 
               
               
                   
               
               
                 41 
                 vlidsvlr 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (1334-1341) 
               
               
                   
               
               
                 42 
                 ilqiitelik 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (1356-1365) 
               
               
                   
               
               
                 43 
                 iigvinehk 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (98-106) 
               
               
                   
               
               
                 44 
                 sleeeekfdpner 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (251-263) 
               
               
                   
               
               
                 45 
                 vvvvagetgsgk 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (590-601) 
               
               
                   
               
               
                 46 
                 vcdelgcengpggr 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (642-655) 
               
               
                   
               
               
                 47 
                 nslcgyqir 
                 Truncated 
               
               
                   
                   
                 peptide from 
               
               
                   
                   
                 DHX29 (656-664) 
               
               
                   
               
               
                 48 
                 acacuugcuuuugacacaacuguguuuacuugcaaucccccaaaacagac 
                 Messenger 
               
               
                   
                 agaauggugcaucuguccagugaggagaagucugcggucacugcccugug 
                 RNA for beta- 
               
               
                   
                 gggcaaggugaauguggaagaaguugguggugaggcccugggcaggcugc 
                 globin 
               
               
                   
                 ugguugucuacccauggacccagagguucuucgaguccuuuggggaccug 
               
               
                   
                 uccucugcaaaugcuguuaugaacaauccuaaggugaaggcucauggcaa 
               
               
                   
                 gaaggugcuggcugccuucagugagggucugagucaccuggacaaccuca 
               
               
                   
                 aaggcaccuuugcuaagcugagugaacugcacugugacaagcugcacgug 
               
               
                   
                 gauccugagaacuucaggcuccugggcaacgugcugguuauugugcuguc 
               
               
                   
                 ucaucauuuuggcaaagaauucacuccucaggugcaggcugccuaucaga 
               
               
                   
                 aggugguggcugguguggccaaugcccuggcucacaaauaccacugagau 
               
               
                   
                 cuuuuucccucugccaaaaauuauggggacaucaugaagccccuugagca 
               
               
                   
                 ucugacuucuggcuaauaaaggaaauuuauuuucauugc 
               
               
                   
               
               
                 49 
                 msasqdsrsr dngpdgmepe gviesnwnei vdsfddmnls esllrgiyay 
                 Eukaryotic 
               
               
                   
                 gfekpsaiqq railpcikgy dviaqaqsgt gktatfaisi lqqieldlka 
                 translation 
               
               
                   
                 tqalvlaptr elaqqiqkvv malgdymgas chaciggtnv raevqklqme 
                 initiation factor 
               
               
                   
                 aphiivgtpg rvfdmlnrry lspkyikmfv ldeademlsr gfkdqiydif 
                 4A isoform 1 
               
               
                   
                 qklnsntqvv llsatmpsdv levtkkfmrd pirilvkkee ltlegirqfy 
                 [ Homo   
               
               
                   
                 invereewkl dtlcdlyetl titqavifin trrkvdwlte kmhardftvs 
                   sapiens ] 
               
               
                   
                 amhgdmdqke rdvimrefrs gssrvlittd llargidvqq vslvinydlp 
               
               
                   
                 tnrenyihri grggrfgrkg vainmvteed krtlrdietf yntsieempl 
               
               
                   
                 nvadli 
               
               
                   
               
               
                 50 
                 maasakkknk kgktisltdf laedggtggg styvskpvsw adetddlegd 
                 Eukaryotic 
               
               
                   
                 vsttwhsndd dvyrappidr silptapraa repnidrsrl pksppytafl 
                 translation 
               
               
                   
                 gnlpydvtee sikeffrgln isavrlprep snperlkgfg yaefedldsl 
                 initiation factor 
               
               
                   
                 lsalslnees lgnrrirvdv adqaqdkdrd drsfgrdrnr dsdktdtdwr 
                 4B [ Homo   
               
               
                   
                 arpatdsfdd ypprrgddsf gdkyrdryds dryrdgyrdg yrdgprrdmd 
                   sapiens ] 
               
               
                   
                 ryggrdrydd rgsrdydrgy dsrigsgrra fgsgyrrddd yrgggdryed 
               
               
                   
                 rydrrddrsw ssrddysrdd yrrddrgppq rpklnlkprs tpkeddssas 
               
               
                   
                 tsqstraasi fggakpvdta arereveerl qkeqeklqrq ldepklerrp 
               
               
                   
                 rerhpswrse etqerersrt gsessqtgts ttssrnarrr esekslenet 
               
               
                   
                 lnkeedchsp tskppkpdqp lkvmpapppk enawvkrssn pparsqssdt 
               
               
                   
                 eqqsptsggg kvapaqpsee gpgrkdenkv dgmnapkgqt 
               
               
                   
                 gnssrgpgdg gnrdhwkesd rkdgkkdqds rsapepkkpe enpaskfssa 
               
               
                   
                 skyaalsvdg edenegedya e 
               
               
                   
               
               
                 51 
                 matvepettp tpnpptteee ktesnqevan pehyikhplq nrwalwffkn 
                 Eukaryotic 
               
               
                   
                 dksktwqanl rliskfdtve dfwalynhiq lssnlmpgcd yslfkdgiep 
                 translation 
               
               
                   
                 mwedeknkrg grwlitlnkq qrrsdldrfw letllclige sfddysddvc 
                 initiation factor 
               
               
                   
                 gavvnvrakg dkiaiwttec enreavthig rvykerlglp pkivigyqsh 
                 4E [ Homo   
               
               
                   
                 adtatksgst tknrfvv 
                   sapiens ] 
               
               
                   
               
               
                 52 
                 mnkapqstgp ppapspglpq pafppgqtap vvfstpqatq mntpsqprqh 
                 Eukaryotic 
               
               
                   
                 fypsraqpps saasrvqsaa parpgpaahv ypagsqvmmi psqisypasq 
                 translation 
               
               
                   
                 gayyipgqgr styvvptqqy pvqpgapgfy pgasptefgt yagayypaqg 
                 initiation factor 
               
               
                   
                 vqqfptgvap apvlmnqppq iapkrerkti rirdpnqggk diteeimsga 
                 4G1 isoform 1 
               
               
                   
                 rtastptppq tggglepqan getpqvaviv rpddrsqgai iadrpglpgp 
                 [ Homo   
               
               
                   
                 ehspsesqps spsptpspsp vlepgsepnl avlsipgdtm ttiqmsvees 
                   sapiens ] 
               
               
                   
                 tpisretgep yrlspeptpl aepilevevt lskpvpesef sssplqaptp 
               
               
                   
                 lashtveihe pngmvpsedl epevesspel apppacpses pvpiaptaqp 
               
               
                   
                 eellngapsp pavdlspvse peeqakevta smapptipsa tpatapsats 
               
               
                   
                 paqeeemeee eeeeegeage ageaesekgg eellppestp ipanlsqnle 
               
               
                   
                 aaaatqvavs vpkrrrkike lnkkeavgdl ldafkeanpa vpevenqppa 
               
               
                   
                 gsnpgpeseg sgvpprpeea detwdskedk ihnaeniqpg 
               
               
                   
                 eqkyeyksdq wkplnleekk rydrefllgf qfifasmqkp eglphisdvv 
               
               
                   
                 ldkanktplr pldptrlqgi ncgpdftpsf anlgrttlst rgpprggpgg 
               
               
                   
                 elprgpaglg prrsqqgprk eprkiiatvl mtediklnka ekawkpsskr 
               
               
                   
                 taadkdrgee dadgsktqdl frrvrsilnk ltpqmfqqlm kqvtqlaidt 
               
               
                   
                 eerlkgvidl ifekaisepn fsvayanmcr clmalkvptt ekptvtvnfr 
               
               
                   
                 klllnrcqke fekdkdddev fekkqkemde aataeergrl keeleeardi 
               
               
                   
                 arrrslgnik figelfklkm lteaimhdcv vkllknhdee sleclcrllt 
               
               
                   
                 tigkdldfek akprmdqyfn qmekiikekk tssrirfmlq dvldlrgsnw 
               
               
                   
                 vprrgdqgpk tidqihkeae meehrehikv qqlmakgsdk rrggppgppi 
               
               
                   
                 srglplvddg gwntvpiskg srpidtsrlt kitkpgsids nnqlfapggr 
               
               
                   
                 lswgkgssgg sgakpsdaas eaarpatstl nrfsalqqav ptestdnrrv 
               
               
                   
                 vqrsslsrer gekagdrgdr lerserggdr gdrldrartp atkrsfskev 
               
               
                   
                 eersrerpsq peglrkaasl tedrdrgrda vkreaalppv splkaalsee 
               
               
                   
                 elekkskaii eeylhlndmk eavqcvqela spsllfifvr hgvestlers 
               
               
                   
                 aiarehmgql lhqllcaghl staqyyqgly eilelaedme idiphvwlyl 
               
               
                   
                 aelvtpilqe ggvpmgelfr eitkplrplg kaasllleil gllcksmgpk 
               
               
                   
                 kvgtlwreag lswkeflpeg qdigafvaeq kveytlgees eapgqralps 
               
               
                   
                 eelnrqlekl lkegssnqrv fdwieanlse qqivsntlvr almtavcysa 
               
               
                   
                 iifetplrvd vavlkarakl lqkylcdeqk elqalyalqa lvvtleqppn 
               
               
                   
                 llrmffdaly dedvvkedaf yswesskdpa eqqgkgvalk svtaffkwlr 
               
               
                   
                 eaeeesdhn 
               
               
                   
               
               
                 53 
                 agcagagtgg cgcagcggaa gcgtgctggg cccataaccc agaggtcgat 
                 Human 
               
               
                   
                 ggatcgaaac catcctctgc tacca 
                 initiator Met- 
               
               
                   
                   
                 tRNA-I 
               
               
                   
               
               
                 54 
                 msaiqnlhsf dpfadaskgd dllpagtedy ihiriqqrng rktlttvqgi 
                 Eukaryotic 
               
               
                   
                 addydkkklv kafkkkfacn gtviehpeyg eviqlqgdqr knicqflvei 
                 translation 
               
               
                   
                 glakddqlkv hgf 
                 initiation factor 1 
               
               
                   
               
               
                 55 
                 mpknkgkggk nrrrgknene sekrelvfke dgqeyaqvik 
                 Eukaryotic 
               
               
                   
                 mlgngrleam cfdgvrrlch irgklrkkvw intsdiilig lrdyqdnkad 
                 translation 
               
               
                   
                 vilkynadea rslkaygelp ehakinetdt fgpgdddeiq fddigddded 
                 initiation factor 
               
               
                   
                 iddi 
                 1A 
               
               
                   
               
               
                 56 
                 mpglscrfyq hkfpevedvv mvnvrsiaem gayvslleyn niegmilise 
                 Eukaryotic 
               
               
                   
                 lsrrrirsin klirigrnec vvvirvdkek gyidlskrrv speeaikced 
                 translation 
               
               
                   
                 kftksktvys ilrhvaevle ytkdeqlesl fqrtawvfdd kykrpgygay 
                 initiation factor 
               
               
                   
                 dafkhavsdp sildsldlne derevlinni nrrltpqavk iradievacy 
                 2, subunit 1 
               
               
                   
                 gyegidavke alraglncst enmpikinli appryvmttt tlerteglsv 
                 alpha [ Homo   
               
               
                   
                 lsqamavike kieekrgvfn vqmepkvvtd tdetelarqm erlerenaev 
                   sapiens ] 
               
               
                   
                 dgdddaeeme akaed 
               
               
                   
               
               
                 57 
                 msgdemifdp tmskkkkkkk kpfmldeegd tqteetqpse tkevepepte 
                 Eukaryotic 
               
               
                   
                 dkdleadeed trkkdasddl ddlnffnqkk kkkktkkifd ideaeegvkd 
                 translation 
               
               
                   
                 lkiesdvqep tepeddldim lgnkkkkkkn vkfpdedeil ekdealeded 
                 initiation factor 
               
               
                   
                 nkkddgisfs nqtgpawags erdytyeell nrvfnimrek npdmvagekr 
                 2 beta [ Homo   
               
               
                   
                 kfvmkppqvv rvgtkktsfv nftdickllh rqpkhllafl laelgtsgsi 
                   sapiens ] 
               
               
                   
                 dgnnqlvikg rfqqkqienv lrryikeyvt chtcrspdti lqkdtrlyfl 
               
               
                   
                 qcetchsrcs vasiktgfqa vtgkraqlra kan 
               
               
                   
               
               
                 58 
                 maggeagvtl gqphlsrqdl ttldvtkltp lshevisrqa tinigtighv 
                 Eukaryotic 
               
               
                   
                 ahgkstvvka isgvhtvrfk nelernitik lgyanakiyk lddpscprpe 
                 translation 
               
               
                   
                 cyrscgsstp defptdipgt kgnfklvrhv sfvdcpghdi lmatmlngaa 
                 initiation factor 
               
               
                   
                 vmdaalllia gnescpqpqt sehlaaieim klkhililqn kidlvkesqa 
                 2, subunit 3 
               
               
                   
                 keqyeqilaf vqgtvaegap iipisaqlky nievvceyiv kkipvpprdf 
                 gamma [ Homo   
               
               
                   
                 tseprlivir sfdvnkpgce vddlkggvag gsilkgvlkv gqeievrpgi 
                   sapiens ] 
               
               
                   
                 vskdsegklm ckpifskivs lfaehndlqy aapggligvg tkidptlcra 
               
               
                   
                 drmvgqvlga vgalpeifte leisyfllrr llgvrtegdk kaakvqklsk 
               
               
                   
                 nevlmvnigs lstggrvsav kadlgkivlt npvctevgek ialsrrvekh 
               
               
                   
                 wrligwgqir rgvtikptvd dd 
               
               
                   
               
               
                 59 
                 mpayfqrpen alkranefle vgkkqpaldv lydvmkskkh rtwqkihepi 
                 Eukaryotic 
               
               
                   
                 mlkylelcvd lrkshlakeg lyqyknicqq vniksledvv raylkmaeek 
                 translation 
               
               
                   
                 teaakeesqq mvldiedldn iqtpesvlls avsgedtqdr tdrllltpwv 
                 initiation factor 
               
               
                   
                 kflwesyrqc ldllrnnsrv erlyhdiaqq afkfclqytr kaefrklcdn 
                 3A [ Homo   
               
               
                   
                 lrmhlsqiqr hhnqstainl nnpesqsmhl etrlvqldsa ismelwqeaf 
                   sapiens ] 
               
               
                   
                 kavedihglf slskkppkpq lmanyynkvs tvfwksgnal fhastlhrly 
               
               
                   
                 hlsremrknl tqdemqrmst rvllatlsip itpertdiar lldmdgiive 
               
               
                   
                 kqrrlatllg lqapptrigl indmvrfnvl qyvvpevkdl ynwlevefnp 
               
               
                   
                 lklcervtkv lnwvreqpek epelqqyvpq lqnntilrll qqvsqiyqsi 
               
               
                   
                 efsrltslvp fvdafqlera ivdaarhcdl qvridhtsrt lsfgsdlnya 
               
               
                   
                 tredapigph lqsmpseqir nqltamssvl akalevikpa hilqekeeqh 
               
               
                   
                 qlavtaylkn srkehqrila rrqtieerke rleslniqre keeleqreae 
               
               
                   
                 lqkvrkaeee rlrqeakere kerilqeheq ikkktvrerl eqikktelga 
               
               
                   
                 kafkdidied leeldpdfim akqveqleke kkelqerlkn qekkidyfer 
               
               
                   
                 akrleeipli ksayeeqrik dmdlweqqee erittmqler ekalehknrm 
               
               
                   
                 srmledrdlf vmrlkaarqs vyeeklkqfe erlaeerhnr leerkrqrke 
               
               
                   
                 errityyrek eeeeqrraee qmlkereere raerakreee lreyqervkk 
               
               
                   
                 leeverkkrq releieerer rreeerrlgd sslsrkdsrw gdrdsegtwr 
               
               
                   
                 kgpeadsewr rgppekewrr gegrdedrsh rrdeerprrl gddedrepsl 
               
               
                   
                 rpdddrvprr gmdddrgprr gpeedrfsrr gadddrpswr ntdddrpprr 
               
               
                   
                 iadedrgnwr hadddrpprr gldedrgswr tadedrgprr gmdddrgprr 
               
               
                   
                 ggadderssw rnadddrgpr rgldddrgpr rgmdddrgpr rgmdddrgpr 
               
               
                   
                 rgmdddrgpr rgldddrgpw rnadddripr rgaeddrgpw rnmdddrlsr 
               
               
                   
                 radddrfprr gddsrpgpwr plvkpggwre kekareeswg ppresrpsee 
               
               
                   
                 rewdrekerd rdnqdreend kdpererdre rdvdredrfr rprdeggwrr 
               
               
                   
                 gpaeessswr dssrrddrdr ddrrrerddr rdlrerrdlr ddrdrrgppl 
               
               
                   
                 rsereevssw rraddrkddr veerdpprrv pppalsrdre rdrdrerege 
               
               
                   
                 kekaswraek dreslrrtkn etdedgwttv rr 
               
               
                   
               
               
                 60 
                 mqdaenvavp eaaeeraepg qqqpaaeppp aegllrpagp gapeaagtea 
                 Eukaryotic 
               
               
                   
                 sseevgiaea gpesevrtep aaeaeaasgp sespsppaae elpgshaepp 
                 translation 
               
               
                   
                 vpaqgeapge qardersdsr aqavsedagg negraaeaep ralengdade 
                 initiation factor 
               
               
                   
                 psfsdpedfv ddvseeellg dvlkdrpqea dgidsvivvd nvpqvgpdrl 
                 3B [ Homo   
               
               
                   
                 eklknvihki fskfgkitnd fypeedgktk gyifleyasp ahavdavkna 
                   sapiens ] 
               
               
                   
                 dgykldkqht frvnlftdfd kymtisdewd ipekqpfkdl gnlrywleea 
               
               
                   
                 ecrdqysvif esgdrtsifw ndvkdpvsie erarwtetyv rwspkgtyla 
               
               
                   
                 tfhqrgialw ggekfkqiqr fshqgvqlid fspcerylvt fsplmdtqdd 
               
               
                   
                 pqaiiiwdil tghkkrgfhc essahwpifk wshdgkffar mtldtlsiye 
               
               
                   
                 tpsmglldkk slkisgikdf swspggniia fwvpedkdip arvtlmqlpt 
               
               
                   
                 rqeirvrnlf nvvdcklhwq kngdylcvkv drtpkgtqgv vtnfeifrmr 
               
               
                   
                 ekqvpvdvve mketiiafaw epngskfavl hgeaprisvs fyhvknngki 
               
               
                   
                 elikmfdkqq antifwspqg qfvvlaglrs mngalafvdt sdctvmniae 
               
               
                   
                 hymasdvewd ptgryvvtsv swwshkvdna ywlwtfqgrl 
               
               
                   
                 lqknnkdrfc qllwrprppt llsqeqikqi kkdlkkyski feqkdrlsqs 
               
               
                   
                 kaskelverr rtmmedfrky rkmaqelyme qknerlelrg gvdtdeldsn 
               
               
                   
                 vddweeetie ffvteeiipl gnqe 
               
               
                   
               
               
                 61 
                 msrffttgsd sesesslsge elvtkpvggn ygkqplllse deedtkrvvr 
                 Eukaryotic 
               
               
                   
                 sakdkrfeel tnlirtirna mkirdvtkcl eefellgkay gkaksivdke 
                 translation 
               
               
                   
                 gvprfyiril adledylnel wedkegkkkm nknnakalst lrqkirkynr 
                 initiation factor 
               
               
                   
                 dfeshitsyk qnpeqsaded aekneedseg ssdedededg vsaatflkkk 
                 3C [ Homo   
               
               
                   
                 seapsgesrk flkkmddede dsedsedded wdtgstssds 
                   sapiens ] 
               
               
                   
                 dseeeegkqt alasrflkka pttdedkkaa ekkredkakk khdrkskrld 
               
               
                   
                 eeeednegge wervrggvpl vkekpkmfak gteithavvi kklneilqar 
               
               
                   
                 gkkgtdraaq iellqllvqi aaennlgegv ivkikfniia slydynpnla 
               
               
                   
                 tymkpemwgk cldcinelmd ilfanpnifv genileesen lhnadqplrv 
               
               
                   
                 rgciltlver mdeeftkimq ntdphsqeyv ehlkdeaqvc 
               
               
                   
                 aiiervqryl eekgtteevc riyllrilht yykfdykahq rqltppegss 
               
               
                   
                 kseqdqaene gedsavlmer lckyiyakdr tdrirtcail chiyhhalhs 
               
               
                   
                 rwyqardlml mshlqdniqh adppvqilyn rtmvqlgica frqgltkdah 
               
               
                   
                 nalldiqssg rakellgqgl llrslqernq eqekverrrq vpfhlhinle 
               
               
                   
                 llecvylvsa mlleipymaa hesdarrrmi skqfhhqlrv gerqpllgpp 
               
               
                   
                 esmrehvvaa skamkmgdwk tchsfiinek mngkvwdlfp 
               
               
                   
                 eadkvrtmlv rkiqeeslrt ylftyssvyd sismetlsdm feldlptvhs 
               
               
                   
                 iiskmiinee lmasldqptq tvvmhrtept aqqnlalqla eklgslvenn 
               
               
                   
                 ervfdhkqgt yggyfrdqkd gyrknegymr rggyrqqqsq tay 
               
               
                   
               
               
                 62 
                 makfmtpviq dnpsgwgpca vpeqfrdmpy qpfskgdrlg 
                 Eukaryotic 
               
               
                   
                 kvadwtgaty qdkrytnkys sqfgggsqya yfheedessf qlvdtartqk 
                 translation 
               
               
                   
                 tayqrnrmrf aqrnlrrdkd rrnmlqfnlq ilpksakqke rerirlqkkf 
                 initiation factor 
               
               
                   
                 qkqfgvrqkw dqksqkprds svevrsdwev keemdfpqlm 
                 3D [ Homo   
               
               
                   
                 kmrylevsep qdieccgale yydkafdrit trsekplrsi krifhtvttt 
                   sapiens ] 
               
               
                   
                 ddpvirklak tqgnvfatda ilatlmsctr svyswdivvq rvgsklffdk 
               
               
                   
                 rdnsdfdllt vsetaneppq degnsfnspr nlameatyin hnfsqqclrm 
               
               
                   
                 gkerynfpnp npfveddmdk neiasvayry rrwklgddid livrcehdgv 
               
               
                   
                 mtgangevsf iniktlnewd srhcngvdwr qkldsqrgav iatelknnsy 
               
               
                   
                 klarwtccal lagseylklg yvsryhvkds srhvilgtqq fkpnefasqi 
               
               
                   
                 nlsvenawgi lrcvidicmk leegkylilk dpnkqvirvy slpdgtfssd 
               
               
                   
                 edeeeeeeee eeeeeeet 
               
               
                   
               
               
                 63 
                 maeydlttri ahfldrhlvf plleflsvke iynekellqg kldllsdtnm 
                 Eukaryotic 
               
               
                   
                 vdfamdvykn lysddiphal rekrttvvaq lkqlqaetep ivkmfedpet 
                 translation 
               
               
                   
                 trqmqstrdg rmlfdyladk hgfrqeyldt lyryakfqye cgnysgaaey 
                 initiation factor 
               
               
                   
                 lyffrvlvpa tdrnalsslw gklaseilmq nwdaamedlt rlketidnns 
                 3E [ Homo   
               
               
                   
                 vssplqslqq rtwlihwslf vffnhpkgrd niidlflyqp qylnaiqtmc 
                   sapiens ] 
               
               
                   
                 philryltta vitnkdvrkr rqvlkdlvkv iqqesytykd pitefvecly 
               
               
                   
                 vnfdfdgaqk klrecesvlv ndfflvacle dfienarlfi fetfcrihqc 
               
               
                   
                 isinmladkl nmtpeeaerw ivnlirnarl dakidsklgh vvmgnnavsp 
               
               
                   
                 yqqviektks lsfrsqmlam niekklnqns rseapnwatq dsgfy 
               
               
                   
               
               
                 64 
                 matpavpvsa ppatptpvpa aapasvpapt papaaapvpa aapasssdpa 
                 Eukaryotic 
               
               
                   
                 aaaaataapg qtpasaqapa qtpapalpgp alpgpfpggr vvrlhpvila 
                 translation 
               
               
                   
                 sivdsyerrn egaarvigtl lgtvdkhsve vtncfsvphn esedevavdm 
                 initiation factor 
               
               
                   
                 efaknmyelh kkvspnelil gwyatghdit ehsvliheyy sreapnpihl 
                 3F [ Homo   
               
               
                   
                 tvdtslqngr msikayvstl mgvpgrtmgv mftpltvkya yydterigvd 
                   sapiens ] 
               
               
                   
                 limktcfspn rviglssdlq qvggasariq dalstvlqya edvlsgkvsa 
               
               
                   
                 dntvgrflms lvnqvpkivp ddfetmlnsn indllmvtyl anltqsqial 
               
               
                   
                 neklvnl 
               
               
                   
               
               
                 65 
                 mptgdfdskp swadqveeeg eddkcvtsel lkgiplatgd tspepellpg 
                 Eukaryotic 
               
               
                   
                 aplpppkevi ngniktvtey kidedgkkfk ivrtfrietr kaskavarrk 
                 translation 
               
               
                   
                 nwkkfgnsef dppgpnvatt tvsddvsmtf itskedlncq eeedpmnklk 
                 initiation factor 
               
               
                   
                 gqkivscric kgdhwttrcp ykdtlgpmqk elaeqlglst gekeklpgel 
                 3G [ Homo   
               
               
                   
                 epvqatqnkt gkyvppslrd gasrrgesmq pnrraddnat irvtnlsedt 
                   sapiens ] 
               
               
                   
                 retdlqelfr pfgsisriyl akdkttgqsk gfafisfhrr edaaraiagv 
               
               
                   
                 sgfgydhlil nvewakpstn 
               
               
                   
               
               
                 66 
                 masrkegtgs tatsssstag aagkgkgkgg sgdsavkqvq idglvvlkii 
                 Eukaryotic 
               
               
                   
                 khyqeegqgt evvqgvllgl vvedrleitn cfpfpqhted dadfdevqyq 
                 translation 
               
               
                   
                 memmrslrhv nidhlhvgwy qstyygsfvt ralldsqfsy qhaieesvvl 
                 initiation factor 
               
               
                   
                 iydpiktaqg slslkayrlt pklmevckek dfspealkka nitfeymfee 
                 3H [ Homo   
               
               
                   
                 vpiviknshl invlmwelek ksavadkhel lslassnhlg knlqllmdrv 
                   sapiens ] 
               
               
                   
                 demsqdivky ntymrntskq qqqkhqyqqr rqqenmqrqs 
               
               
                   
                 rgepplpeed lsklfkppqp parmdsllia gqintycqni keftaqnlgk 
               
               
                   
                 lfmaqalqey nn 
               
               
                   
               
               
                 67 
                 mkpillqghe rsitqikynr egdllftvak dpivnvwysv ngerlgtymg 
                 Eukaryotic 
               
               
                   
                 htgavwcvda dwdtkhvltg sadnscrlwd cetgkqlall ktnsavrtcg 
                 translation 
               
               
                   
                 fdfggniimf stdkqmgyqc fvsffdlrdp sqidnnepym kipcndskit 
                 initiation factor 
               
               
                   
                 savwgplgec iiaghesgel nqysaksgev lvnvkehsrq indiqlsrdm 
                 3I [ Homo   
               
               
                   
                 tmfvtaskdn taklfdsttl ehqktfrter pvnsaalspn ydhvvlgggq 
                   sapiens ] 
               
               
                   
                 eamdvtttst rigkfearff hlafeeefgr vkghfgpins vafhpdgksy 
               
               
                   
                 ssggedgyvr ihyfdpqyfe fefea 
               
               
                   
               
               
                 68 
                 maaaaaaagd sdswdadafs vedpvrkvgg ggtaggdrwe 
                 Eukaryotic 
               
               
                   
                 gedededvkd nwdddddekk eeaevkpevk isekkkiaek 
                 translation 
               
               
                   
                 ikekerqqkk rqeeikkrle epeepkvltp eeqladklrl kklqeesdle 
                 initiation factor 
               
               
                   
                 laketfgvnn avygidamnp ssrddftefg kllkdkitqy ekslyyasfl 
                 3J [ Homo   
               
               
                   
                 evlvrdvcis leiddlkkit nsltvlcsek qkqekqskak kkkkgvvpgg 
                   sapiens ] 
               
               
                   
                 glkatmkddl adyggydggy vqdyedfm 
               
               
                   
               
               
                 69 
                 mamfeqmran vgkllkgidr ynpenlatle ryvetqaken aydleanlav 
                 Eukaryotic 
               
               
                   
                 lklyqfnpaf fqttvtaqil lkaltnlpht dftlckcmid qahqeerpir 
                 translation 
               
               
                   
                 qilylgdlle tchfqafwqa ldenmdlleg itgfedsvrk fichvvgity 
                 initiation factor 
               
               
                   
                 qhidrwllae mlgdlsdsql kvwmskygws adesgqific sqeesikpkn 
                 3K [ Homo   
               
               
                   
                 ivekidfdsv ssimassq 
                   sapiens ] 
               
               
                   
               
               
                 70 
                 msypaddyes eaaydpyayp sdydmhtgdp kqdlayerqy 
                 Eukaryotic 
               
               
                   
                 eqqtyqvipe viknfiqyfh ktvsdlidqk vyelqasrvs sdvidqkvye 
                 translation 
               
               
                   
                 iqdiyenswt klterffknt pwpeaeaiap qvgndavfli lykelyyrhi 
                 initiation factor 
               
               
                   
                 yakvsggpsl eqrfesyyny cnlfnyilna dgpaplelpn qwlwdiidef 
                 3L [ Homo   
               
               
                   
                 iyqfqsfsqy rcktakksee eidflrsnpk iwnvhsvlnv lhslvdksni 
                   sapiens ] 
               
               
                   
                 nrqlevytsg gdpesvagey grhslykmlg yfslvgllrl hsllgdyyqa 
               
               
                   
                 ikvlenieln kksmysrvpe cqvttyyyvg faylmmrryq dairvfanil 
               
               
                   
                 lyiqrtksmf qrttykyemi nkqneqmhal laialtmypm ridesihlql 
               
               
                   
                 rekygdkmlr mqkgdpqvye elfsyscpkf lspvvpnydn 
               
               
                   
                 vhpnyhkepf lqqlkvfsde vqqqaqlsti rsflklyttm pvaklagfld 
               
               
                   
                 lteqefriql lvfkhkmknl vwtsgisald gefqsasevd fyidkdmihi 
               
               
                   
                 adtkvarryg dffirqihkf eelnrtlkkm gqrp 
               
               
                   
               
               
                 71 
                 msvpafidis eedqaaelra ylkskgaeis eensegglhv dlaqiieacd 
                 Eukaryotic 
               
               
                   
                 vclkeddkdv esvmnsvvsl llilepdkqe alieslcekl vkfregerps 
                 translation 
               
               
                   
                 lrlqllsnlf hgmdkntpvr ytvycslikv aascgaiqyi pteldqvrkw 
                 initiation factor 
               
               
                   
                 isdwnlttek khtllrllye alvdckksda askvmvellg sytednasqa 
                 3M [ Homo   
               
               
                   
                 rvdahrcivr alkdpnaflf dhlltlkpvk flegelihdl ltifvsakla 
                   sapiens ] 
               
               
                   
                 syvkfyqnnk dfidslgllh eqnmakmrll tfmgmavenk eisfdtmqqe 
               
               
                   
                 lqigaddvea fvidavrtkm vyckidqtqr kvvvshsthr tfgkqqwqql 
               
               
                   
                 ydtlnawkqn lnkvknslls lsdt 
               
               
                   
               
               
                 72 
                 ggaauucaacgcagaguacgcggggcaacacugagaaguuaucuuaaggg 
                 (NCF2) 5′- 
               
               
                   
                 aggcugggccccauucuacucaucuggcccagaaagugaacaccuugggg 
                 UTR 
               
               
                   
                 gccacuaaggcagcccugcuaggggagacgcuccaaccugucuucucucu 
               
               
                   
                 gucuccuggcagcucucuuggccuccuaguuucuaccuaauccauggaag 
               
               
                   
                 acgccaaaaacauaaagaaaggcccggcgccauucuauccgcuggaagau 
               
               
                   
                 ggaaccgcuggagagcaacugcauaaggcuaugaagagauacgcccuggu 
               
               
                   
                 uccuggaacaauugcuuuuacagaugcacauaucgagguggacaucacuu 
               
               
                   
                 acgcugaguacuucgaaauguccguucgguuggcagaagcuaugaaacga 
               
               
                   
                 uaugggcugaauacaaaucacagaaucgucguaugcagugaaaacucucu 
               
               
                   
                 ucaauucuuuaugccgguguugggcgcguuauuuaucggaguugcaguug 
               
               
                   
                 cgcccgcgaacgacauuuauaaugaacgugaauugcucaacaguaugggc 
               
               
                   
                 auuucgcagccuaccgugguguucguuuccaaaaagggguugcaaaaaau 
               
               
                   
                 uuugaacgugcaaaaaaagcucccaaucauccaaaaaauuauuaucaugg 
               
               
                   
                 auucuaaaacggauuaccagggauuucagucgauguacacguucgucaca 
               
               
                   
                 ucucaucuaccucccgguuuuaaugaauacgauuuugugccagaguccuu 
               
               
                   
                 cgauagggacaagacaauugcacugaucaugaacuccucuggaucuacug 
               
               
                   
                 gucugccuaaaggugucgcucugccucauagaacugccugcgugagauuc 
               
               
                   
                 ucgcaugccagagauccuauuuuuggcaaucaaaucauuccggauacugc 
               
               
                   
                 gauuuuaaguguuguuccauuccaucacgguuuuggaauguuuacuacac 
               
               
                   
                 ucggauauuugauauguggauuucgagucgucuuaauguauagauuugaa 
               
               
                   
                 gaagagcuguuucugaggagccuucaggauuacaagauucaaagugcgcu 
               
               
                   
                 gcuggugccaacccuauucuccuucuucgccaaaagcacucugauugaca 
               
               
                   
                 aauacgauuuaucuaauuuacacgaaauugcuucugguggcgcuccccuc 
               
               
                   
                 ucuaaggaagucggggaagcgguugccaagagguuccaucugccagguau 
               
               
                   
                 caggcaaggauaugggcucacugagacuacaucagcuauucugauuacac 
               
               
                   
                 ccgagggggaugauaaaccgggcgcggucgguaaaguuguuccauuuuuu 
               
               
                   
                 gaagcgaagguuguggaucuggauaccgggaaaacgcugggcguuaauca 
               
               
                   
                 aagaggcgaacugugugugagagguccuaugauuauguccgguuauguaa 
               
               
                   
                 acaauccggaagcgaccaacgccuugauugacaaggauggauggcuacau 
               
               
                   
                 ucuggagacauagcuuacugggacgaagacgaacacuucuucaucguuga 
               
               
                   
                 ccgccugaagucucugauuaaguacaaaggcuaucagguggcucccgcug 
               
               
                   
                 aauuggaauccaucuugcuccaacaccccaacaucuucgacgcagguguc 
               
               
                   
                 gcaggucuucccgacgaugacgccggugaacuucccgccgccguuguugu 
               
               
                   
                 uuuggagcacggaaagacgaugacggaaaaagagaucguggauuacgucg 
               
               
                   
                 ccagucaaguaacaaccgcgaaaaaguugcgcggaggaguuguguuugug 
               
               
                   
                 gacgaaguaccgaaaggucuuaccggaaaacucgacgcaagaaaaaucag 
               
               
                   
                 agagauccucauaaaggccaagaagggcggaaagaucgccguguaauucu 
               
               
                   
                 agagucggggcggccggccgcuucgagcagacaugauaagauacauugau 
               
               
                   
                 gaguuuggacaaaccacaacuagaaugcagugaaaaaaaugcuuuauuug 
               
               
                   
                 ugaaauuugugaugcuauugcuuuauuuguaaccauuauaagcugcaaua 
               
               
                   
                 aacaaguuaacaacaacaauugcauucauuuuauguuucagguucagggg 
               
               
                   
                 gaggugugggagguuuuuuaaagcaaguaaaaccucuacaaaugugguaa 
               
               
                   
                 aaucgauaaggaucugaacgauggagcggagaaugggcggaacugggcgg 
               
               
                   
                 aguuaggggcgggaugggcggaguuaggggcgggacuaugguugcugacu 
               
               
                   
                 aauugagaugcaugcuuugcauacuucugccugcuggggagccuggggac 
               
               
                   
                 uuuccacaccugguugcugacuaauugagaugcaugcuuugcauacuucu 
               
               
                   
                 gccugcuggggagccuggggacuuuccacacccuaacugacacacauucc 
               
               
                   
                 acagcggauc 
               
               
                   
               
               
                 73 
                 gggcggccgcgaauucggucaacgccugcggcuguugauauucuugcuca 
                 CDC25 mRNA 
               
               
                   
                 gaggccguaacuuuggccuucugcucagggaagacucugaguccgacguu 
               
               
                   
                 ggccuacccagucggaaggcagagcugcaaucuaguuaacuaccuccuuu 
               
               
                   
                 ccccuagauuuccuuucauucugcucaagucuucgccuguguccgauccc 
               
               
                   
                 uaucuacuuucucuccucuuguaggcaagccucagacuccaggcuugagc 
               
               
                   
                 uagguuuuguuuuucuccuggugagaauucgaagaccaugucuacggaac 
               
               
                   
                 ucuucucauccacaagagaggaaggaagcucuggcucaggacccaguuuu 
               
               
                   
                 aggucuaaucaaaggaaaauguuaaaccugcuccuggagagagacacuuc 
               
               
                   
                 cuuuaccgucuguccagaugucccuagaacuccagugggcaaauuucuug 
               
               
                   
                 gugauucugcaaaccuaagcauuuugucuggaggaaccccaaaacguugc 
               
               
                   
                 cucgaucuuucgaaucuuagcaguggggagauaacugccacucagcuuac 
               
               
                   
                 cacuucugcagaccuugaugaaacuggucaccuggauucuucaggacuuc 
               
               
                   
                 aggaagugcauuuagcugggaugaaucaugaccagcaccuaaugaaaugu 
               
               
                   
                 agcccagcacagcuucuuuguagcacuccgaaugguuuggaccguggcca 
               
               
                   
                 uagaaagagagaugcaauguguaguucaucugcaaauaaagaaaaugaca 
               
               
                   
                 auggaaacuugguggacagugaaaugaaauauuugggcagucccauuacu 
               
               
                   
                 acuguuccaaaauuggauaaaaauccaaaccuaggagaagaccaggcaga 
               
               
                   
                 agagauuucagaugaauuaauggaguuuucccugaaagaucaagaagcaa 
               
               
                   
                 aggugagcagaaguggccuauaucgcuccccgucgaugccagagaacuug 
               
               
                   
                 aacaggccaagacugaagcagguggaaaaauucaaggacaacacaauacc 
               
               
                   
                 agauaaaguuaaaaaaaaguauuuuucuggccaaggaaagcucaggaagg 
               
               
                   
                 gcuuauguuuaaagaagacagucucucugugugacauuacuaucacucag 
               
               
                   
                 augcuggaggaagauucuaaccaggggcaccugauuggugauuuuuccaa 
               
               
                   
                 gguaugugcgcugccaaccgugucagggaaacaccaagaucugaaguaug 
               
               
                   
                 ucaacccagaaacaguggcugccuuacugucggggaaguuccagggucug 
               
               
                   
                 auugagaaguuuuaugucauugauugucgcuauccauaugaguaucuggg 
               
               
                   
                 aggacacauccagggagccuuaaacuuauauagucaggaagaacuguuua 
               
               
                   
                 acuucuuucugaagaagcccaucgucccuuuggacacccagaagagaaua 
               
               
                   
                 aucaucguguuccacugugaauucuccucagagaggggcccccgaaugug 
               
               
                   
                 ccgcugucugcgugaagaggacaggucucugaaccaguauccugcauugu 
               
               
                   
                 acuacccagagcuauauauccuuaaaggcggcuacagagacuucuuucca 
               
               
                   
                 gaauauauggaacugugugaaccacagagcuacugcccuaugcaucauca 
               
               
                   
                 ggaccacaagacugaguugcugaggugucgaagccagagcaaagugcagg 
               
               
                   
                 aaggggagcggcagcugcgggagcagauugcccuucuggugaaggacaug 
               
               
                   
                 agcccaugauaacauuccagccacuggcugcuaacaagucaccaaaagac 
               
               
                   
                 acugcagaaacccugagcagaaagaggccuucuggauggccaaacccaag 
               
               
                   
                 auuauuaaaagaugucucugcaaaccaacaggcuaccaacuuguauccag 
               
               
                   
                 gccugggaauggauuagguuucagcagagcugaaagcugguggcagaguc 
               
               
                   
                 cuggagcuggcucuauaaggcagccuugaguugcauagagauuuguauug 
               
               
                   
                 guucagggaacucuggcauuccuuuucccaacuccucaugucuucucaca 
               
               
                   
                 agccagccaacucuuucucucugggcuucgggcuaugcaagagcguuguc 
               
               
                   
                 uaccuucuuucuuuguauuuuccuucuuuguuucccccucuuucuuuuuu 
               
               
                   
                 aaaaauggaaaaauaaacacuacagaaugaggucga 
               
               
                   
               
               
                 74 
                 gcaacaacaacaacaacaacaacaacaacaacaacaacaacaagggcugc 
                 CAA(Stem 3)- 
               
               
                   
                 gggccuccgcagccccaacaacaaccaugguccguccuguagaaacccca 
                 GUS mRNA 
               
               
                   
                 acccgugaaaucaaaaaacucgacggccugugggcauucagucuggaucg 
               
               
                   
                 cgaaaacuguggaauugaucagcguuggugggaaagcgcguuacaagaaa 
               
               
                   
                 gccgggcaauugcugugccaggcaguuuuaacgaucaguucgccgaugca 
               
               
                   
                 auucguaauuaugcgggcaacgucugguaucagcgcgaagucuuuauacc 
               
               
                   
                 gaugaaagguugggcaggccagcguaucgugcugcguuucgaugcgguca 
               
               
                   
                 cucauuacggcaaagugugggucaauaaucaggaagugauggagcaucag 
               
               
                   
                 ggcggcuauacgccauuugaagccgaugucacgccguauguuauugccgg 
               
               
                   
                 gaaaaguguac 
               
               
                   
               
               
                 75 
                 gcaacaacaacaacaacaacaacaacaacaacaacaacaacaagggcugc 
                 CAA(Stem 4)- 
               
               
                   
                 gguggagccuuccaccgcagccccaacaacaaccaugguccguccuguag 
                 GUS mRNA 
               
               
                   
                 aaaccccaacccgugaaaucaaaaaacucgacggccugugggcauucagu 
               
               
                   
                 cuggaucgcgaaaacuguggaauugaucagcguuggugggaaagcgcguu 
               
               
                   
                 acaagaaagccgggcaauugcugugccaggcaguuuuaacgaucaguucg 
               
               
                   
                 ccgaugcaauucguaauuaugcgggcaacgucugguaucagcgcgaaguc 
               
               
                   
                 uuuauaccgaugaaagguugggcaggccagcguaucgugcugcguuucga 
               
               
                   
                 ugcggucacucauuacggcaaagugugggucaauaaucaggaagugaugg 
               
               
                   
                 agcaucagggcggcuauacgccauuugaagccgaugucacgccguauguu 
               
               
                   
                 auugccgggaaaaguguac 
               
               
                   
               
               
                 76 
                 gcaagaacaacaacaacaacaacaacaacaacaacaacaacaacaacaac 
                 (CAA)n-GUS 
               
               
                   
                 aacaacaacaacaacaccaugguccguccuguagaaaccccaacccguga 
                 mRNA 
               
               
                   
                 aaucaaaaaacucgacggccugugggcauucagucuggaucgcgaaaacu 
               
               
                   
                 guggaauugaucagcguuggugggaaagcgcguuacaagaaagccgggca 
               
               
                   
                 auugcugugccaggcaguuuuaacgaucaguucgccgaugcagauauucg 
               
               
                   
                 uaauuaugcgggcaacgucugguaucagcgcgaagucuuuauaccgaaag 
               
               
                   
                 guugggcaggccagcguaucgugcugcguuucgaugcggucacucauuac 
               
               
                   
                 ggcaaagugugggucaauaaucaggaagugauggagcaucagggcggcua 
               
               
                   
                 uacgccauuugaagccgaugucacgccguauguuauugccgggaaaagug 
               
               
                   
                 uac 
               
               
                   
               
               
                 77 
                 gcaacaacaacaacaacaacaacaacaacaacaacaacaacaagggcgcc 
                 CAA(Stem 1)- 
               
               
                   
                 ugccccaacaacaaccaugguccguccuguagaaaccccaacccgugaaa 
                 GUS mRNA 
               
               
                   
                 ucaaaaaacucgacggccugugggcauucagucuggaucgcgaaaacugu 
               
               
                   
                 ggaauugaucagcguuggugggaaagcgcguuacaagaaagccgggcaau 
               
               
                   
                 ugcugugccaggcaguuuuaacgaucaguucgccgaugcagauauucgua 
               
               
                   
                 auuaugcgggcaacgucugguaucagcgcgaagucuuuauaccgaaaggu 
               
               
                   
                 ugggcaggccagcguaucgugcugcguuucgaugcggucacucauuacgg 
               
               
                   
                 caaagugugggucaauaaucaggaagugauggagcaucagggcggcuaua 
               
               
                   
                 cgccauuugaagccgaugucacgccguauguuauugccgggaaaaguguac 
               
               
                   
               
               
                 78 
                 gcaacaacaacaacaacaacaacaacaacaacaacaacaacaagggcugc 
                 CAA(Stem 2)- 
               
               
                   
                 gccugcagccccaacaacaaccaugguccguccuguagaaaccccaaccc 
                 GUS mRNA 
               
               
                   
                 gugaaaucaaaaaacucgacggccugugggcauucagucuggaucgcgaa 
               
               
                   
                 aacuguggaauugaucagcguuggugggaaagcgcguuacaagaaagccg 
               
               
                   
                 ggcaauugcugugccaggcaguuuuaacgaucaguucgccgaugcagaua 
               
               
                   
                 uucguaauuaugcgggcaacgucugguaucagcgcgaagucuuuauaccg 
               
               
                   
                 aaagguugggcaggccagcguaucgugcugcguuucgaugcggucacuca 
               
               
                   
                 uuacggcaaagugugggucaauaaucaggaagugauggagcaucagggcg 
               
               
                   
                 gcuauacgccauuugaagccgaugucacgccguauguuauugccgggaaa 
               
               
                   
                 aguguac 
               
               
                   
               
               
                 79 
                 gcaacaacaacaacaacaacaacaacaacaacaacaacaacaacaaacca 
                 (CAA)N- 
               
               
                   
                 ugcaacaacaaaccaugguccguccuguagaaaccccaacccgugaaauc 
                 AUG-AUG- 
               
               
                   
                 aaaaaacucgacggccugugggcauucagucuggaucgcgaaaacugugg 
                 GUS mRNA 
               
               
                   
                 aauugaucagcguuggugggaaagcgcguuacaagaaagccgggcaauug 
               
               
                   
                 cugugccaggcaguuuuaacgaucaguucgccgaugcagauauucguaau 
               
               
                   
                 uaugcgggcaacgucugguaucagcgcgaagucuuuauaccgaaagguug 
               
               
                   
                 ggcaggccagcguaucgugcugcguuucgaugcggucacucauuacggca 
               
               
                   
                 aagugugggucaauaaucaggaagugauggagcaucagggcggcuauacg 
               
               
                   
                 ccauuugaagccgaugucacgccguauguuauugccgggaaaaguguac 
               
               
                   
               
               
                 80 
                 gggcgaauugggcccucuagaugcaugcucgagcggccgccagugugaug 
                 CrPV IGR 
               
               
                   
                 gauaucuauguuggcugaugagguuuacgacuucuaaaaagcaaaaaugu 
                 IRES 
               
               
                   
                 gaucuugcuuguaaauacaauuuugagagguuaauaaauuacaaguagug 
               
               
                   
                 cuauuuuuguauuuagguuagcuauuuagcuuuacguuccaggaugccua 
               
               
                   
                 guggcagccccacaauauccaggaagcccucucugcgguuuuucagauua 
               
               
                   
                 gguagucgaaaaaccuaagaaauuuaccugcuacauuucaagauaaacaa 
               
               
                   
                 gaaaauucacacauugaaaaugaagauaaaagacuuauguccgaacagaa 
               
               
                   
                 agaaauuguacauuuuguuagcgaaggaauuaccccuaguacuacugcgc 
               
               
                   
                 ucccugauaucguuaaucuuucaacuaauuaucuggacaugacuacgaga 
               
               
                   
                 gaagauagaauu 
               
               
                   
               
               
                 81 
                 gggagaccggaagcugucgacaagguuagcucuuucucguauacgauauu 
                 CSFVΔ442.NS′ 
               
               
                   
                 ggauacacuaaauuucgauuuggucuagggcaccccuccagcgacggccg 
                 mRNA 
               
               
                   
                 aaaugggcuagccaugcccauaguaggacuagcaaacggagggacuagcc 
               
               
                   
                 guaguggcgagcucccuggguggucuaaguccugaguacaggacagucgu 
               
               
                   
                 caguaguucgacgugagcacuagcccaccucgagaugcuacguggacgag 
               
               
                   
                 ggcaugcccaagacacaccuuaacccuggcgggggucgcuagggugaaau 
               
               
                   
                 cacauuaugugauggggguacgaccugauagggugcugcagaggcccacu 
               
               
                   
                 agcaggcuaguauaaaaaucucugcuguacauggcacauggaguugaauc 
               
               
                   
                 auuuugaauuauuauacaaaacaagcaaacaaaaaccagugggaguggag 
               
               
                   
                 gaaccggguaccauggaucccagcuuucagguagauugcuuucuuuggca 
               
               
                   
                 uguccgcaaacgaguugcagaccaagaacuaggugaugccccauuccuug 
               
               
                   
                 aucggcuucgccgagaucagaaaucccuaagaggaaggggcagcacucuu 
               
               
                   
                 ggucuggacaucgagacagccacacgugcuggaaagcagauaguggagcg 
               
               
                   
                 gauucugaaagaagaauccgaugaggcacuuaaaaugaccauggccucug 
               
               
                   
                 uaccugcgucgcguuaccuaaccgacaugacucuugaggaaaugucaagg 
               
               
                   
                 gaaugguccaugcucauacccaagcagaaaguggcaggcccucuuuguau 
               
               
                   
                 cagaauggaccaggcgaucauggauaaaaacaucauacugaaagcgaacu 
               
               
                   
                 ucagugugauuuuugaccggcuggagacucuaauauugcuaagggcuuuc 
               
               
                   
                 accgaagagggagcaauuguuggcgaaauuucaccauugccuucucuucc 
               
               
                   
                 aggacauacugcugaggaugucaaaaaugcaguuggaguccucaucggag 
               
               
                   
                 gacuugaauggaaugauaacacaguucgagucucugaaacucuacagaga 
               
               
                   
                 uucgcuuggagaagcaguaaugagaaugggagaccuccacucacuccaaa 
               
               
                   
                 acagaaacgagaaauggcgggaacaauuaggucagaaguuuugaagaaau 
               
               
                   
                 aagaugguugauugaagaagugagacacaaacugaagguaacagagaaua 
               
               
                   
                 guuuugagcaaauaacauuuaugcaagccuuacaucuauugcuugaagug 
               
               
                   
                 gagcaagagauaagaacuuucucauuucagcuuauuuaauaauaaaaaac 
               
               
                   
                 acccuuguuucuacugaaauu 
               
               
                   
               
               
                 82 
                 gggagaccggaagcugucgacuagccguaguggcgagcucccuggguggu 
                 CSFV(128-442). 
               
               
                   
                 cuaaguccugaguacaggacagucgucaguaguucgacgugagcacuagc 
                 NS′ 
               
               
                   
                 ccaccucgagaugcuacguggacgagggcaugcccaagacacaccuuaac 
                 mRNA 
               
               
                   
                 ccuggcgggggucgcuagggugaaaucacauuaugugauggggguacgac 
               
               
                   
                 cugauagggugcugcagaggcccacuagcaggcuaguauaaaaaucucug 
               
               
                   
                 cuguacauggcacauggaguugaaucauuuugaauuauuauacaaaacaa 
               
               
                   
                 gcaaacaaaaaccagugggaguggaggaaccggguaccauggaucccagc 
               
               
                   
                 uuucagguagauugcuuucuuuggcauguccgcaaacgaguugcagacca 
               
               
                   
                 agaacuaggugaugccccauuccuugaucggcuucgccgagaucagaaau 
               
               
                   
                 cccuaagaggaaggggcagcacucuuggucuggacaucgagacagccaca 
               
               
                   
                 cgugcuggaaagcagauaguggagcggauucugaaagaagaauccgauga 
               
               
                   
                 ggcacuuaaaaugaccauggccucuguaccugcgucgcguuaccuaaccg 
               
               
                   
                 acaugacucuugaggaaaugucaagggaaugguccaugcucauacccaag 
               
               
                   
                 cagaaaguggcaggcccucuuuguaucagaauggaccaggcgaucaugga 
               
               
                   
                 uaaaaacaucauacugaaagcgaacuucagugugauuuuugaccggcugg 
               
               
                   
                 agacucuaauauugcuaagggcuuucaccgaagagggagcaauuguuggc 
               
               
                   
                 gaaauuucaccauugccuucucuuccaggacauacugcugaggaugucaa 
               
               
                   
                 aaaugcaguuggaguccucaucggaggacuugaauggaaugauaacacag 
               
               
                   
                 uucgagucucugaaacucuacagagauucgcuuggagaagcaguaaugag 
               
               
                   
                 aaugggagaccuccacucacuccaaaacagaaacgagaaauggcgggaac 
               
               
                   
                 aauuaggucagaaguuuugaagaaauaagaugguugauugaagaagugag 
               
               
                   
                 acacaaacugaagguaacagagaauaguuuugagcaaauaacauuuaugc 
               
               
                   
                 aagccuuacaucuauugcuugaaguggagcaagagauaagaacuuucuca 
               
               
                   
                 uuucagcuuauuuaauaauaaaaaacacccuuguuucuacugaaauu