Abstract:
The invention is directed to RNA molecules that can be used to inhibit protein synthesis and to induce cells to undergo apoptosis. It also includes pharmaceutical compositions containing the RNAs that can be used in treating or preventing tumors; abnormal dermatological growths and viral infections.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is US national stage of international application PCT/US2009/002718, which had an international filing date of May 1, 2009 and claims the benefit of U.S. provisional application 61/071,522, filed on May 2, 2008, which is incorporated herein by reference in its entirety. The PCT application was published in English under PCT Article 21(2) on Nov. 5, 2009. 
    
    
     STATEMENT OF GOVERNMENT FUNDING 
     This invention was made with Government support under Grant Nos. AI065858, AR051472, and AI033600 awarded by the National Institutes of Health. The Government has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention is directed to short RNA molecules that can be used to inhibit protein synthesis and induce cellular apoptosis. These RNAs may be used in studying posttranscriptional processes involved in controlling gene expression and in therapies designed to kill tumor cells or inhibit virus growth. 
     BACKGROUND OF THE INVENTION 
     Among the most significant discoveries of the last decade was that of RNA-based mechanisms of post-transcriptionally regulating gene expression (Hamilton, et al.,  Nature  431:371-378 (2004); Fire, et al., 391:806-811 (1998); Zamor, et al.,  Cell  101:25-33 (2000). It has been found that, when double stranded RNA enters cells, e.g., due to viral infection, it is recognized and cleaved by specific “dicer enzymes” into fragments (siRNAs) 21-25 nucleotides in length with 3′ dinucleotide overhangs. These fragments bind to a protein complex (RISC) that causes the RNA to unwind and one of the strands, the passenger strand, to be degraded. The remaining sequence targets the complex to an mRNA having a complementary sequence which is then cleaved. As a result, the expression of protein encoded by that mRNA is prevented. 
     Primitive cells and organisms may have used this system to protect themselves from virus infection long before the development of adaptive immune systems. Higher organisms appear to have adapted the system to modulating gene expression. In humans for example, endogenous genomic sequences transcribe RNA capable of folding back on itself to form double stranded regions. These are cleaved by enzymes to form “miRNA” fragments that then act in essentially the same manner as siRNAs. However, unlike siRNAs, the miRNAs often contain mismatches that do not allow the cleavage of mRNA targets. Rather, partially complementary target mRNAs are deadenylated, decapped, and degraded by the 5′-3′ exonucleolytic pathway. Alternatively, these mRNAs can be subject to translational silencing by a mechanism that is poorly characterized (for overview see, Ambros,  Nature  431:350-355 (2004); Mattick,  EMBO Reports  2:986-991 (2001); Bentwick, et al.,  Nature Genetics  37:766-770 (2005)). 
     The process of catalytically shutting down the translation of specific mRNAs by introducing double stranded RNA into cells can be used to target essentially any chosen target transcript. Thus, RNA interference is a technique of great interest clinically (where genes associated with diseases may be targeted) and to researchers attempting to identify the function of genes, e.g., resulting from the human genome project (see generally U.S. Pat. Nos. 7,232,806; 7,078,196 and 7,056,704). 
     Posttranscriptional regulation of gene expression plays an especially important role in the survival of mammalian cells exposed to adverse environmental conditions (Ron, et al.,  Nature reviews  8:519-529 (2007)). Control is accomplished by the downregulation of protein synthesis due to the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (Hershey,  Annu. Rev. Biochem.  60:717-755 (1991)). In addition, recent evidence has indicated the existence of a separate, phospho-eIF2α independent, translation control pathway (McEwen, et al.,  J. Biol. Chem.  280:16925-16933 (2005); Tenson, et al.,  Mol. Microbiol.  59:1664-1677 (2006); Rocha, et al.,  Food Add. Contam.  22:369-378 (2005); Iordanov, et al.,  J. Biol. Chem.  273:15794-15803 (1998); Shifrin, et al.,  J. Biol. Chem.  274:13985-13992 (1999)). 
     In Tetrahymena thermophila, nutrient stress induces cleavage of the tRNA anticodon loop to produce RNA fragments derived from the 5′ and 3′ ends of most, if not all, tRNAs (Lee, et al.,  J. Biol. Chem.  280:42744-42749 (2005)). The 3′ fragments of these tiRNAs lack the terminal CCA residues required for aminoacylation, suggesting that anticodon cleavage occurs following 3′ end processing, but prior to CCA addition. In mammalian cells, analogous RNAs comprise a small subset of piwi-associated piRNAs suggesting that tRNA anticodon cleavage may be a widespread phenomenon that can lead to the assembly of specific RNP complexes (Brennecke, et al.,  Cell  128:1089-1103 (2007); Grivna, et al.,  Proc. Nat&#39;l Acad. Sci. USA  103:13415-13420 (2006); Lau, et al., Science 313:363-367 (2006)). 
     SUMMARY OF THE INVENTION 
     The present invention is based upon the discovery that, in response to stress, cells cleave tRNAs in or near the anticodon loop to form fragments (referred to herein as “tiRNAs” or, depending on context, more simply as “RNAs”) that inhibit the translation of all mRNA transcripts. Cells treated with tiRNAs were found to undergo apoptosis. Thus, locally delivered synthetic RNAs corresponding to tiRNAs can be used to kill cells for either scientific or therapeutic purposes. 
     In its first aspect, the invention is directed to a method of inhibiting protein synthesis or inducing apoptosis in a population of cells by administering an effective amount of an RNA molecule 18-35, and preferably 28-30, nucleotides in length. These RNAs must have a sequence that is at least 90% identical to a sequence found within the first 40 nucleotides at the 5′ end of a human tRNA. The term “effective amount” or “therapeutically effective amount” refers to a sufficient amount of tiRNA to inhibit protein synthesis by at least 10% and/or induce apoptosis in at least 10% of the cells treated and preferably in or by 20, 40, 60 or 80%. 
     The RNA may be part of a solution, suspension, or emulsion in which it is present at a concentration of 1 ng/ml-100 μg/ml, and preferably at a concentration of 10 ng/ml-10 μg/ml. Cells should typically be contacted with 10 ng-1 mg (and preferably 1 μg-500 μg of the RNA per million cells) and may be found either in vitro, e.g., growing in culture, or in vivo. For example, the RNA may be used to treat benign or malignant tumors, abnormal growths (especially melanomas) on the skin of a subject or conditions such as macular degeneration in which normal cells grow excessively. Most preferably, the RNA molecule is at least 18 (and preferably 28-30) nucleotides long and at least 90% (and preferably 100%) identical to a sequence in SEQ ID NO:1-SEQ ID NO:132 as shown in Table 1. 
     In another aspect, the invention is directed to a topical composition in the form of a solution, cream, lotion, suspension, emulsion or gel, comprising 0.0001-10 weight percent (and preferably 0.001-1 wt %) of an RNA molecule 18-35 (and preferably 28-30) nucleotides in length. The RNA should comprise a sequence at least 18 nucleotides long and be at least 90% identical to a sequence in SEQ ID NO:1-SEQ ID NO:132 as shown in Table 1 together with one or more excipients. The RNA should be present in the composition at a concentration of 1 ng/ml-100 μg/ml, and preferably 10 ng/ml-10 μg/ml. The topical composition will be useful in treating skin conditions characterized by abnormal cellular growth such a melanomas. 
     The invention also includes injectable pharmaceutical compositions comprising 0.0001-10 weight percent (wt %) of RNA molecules with the characteristics described above together with a sterile carrier. These compositions may be delivered to sites of virus infection or applied as a microbicide to prevent virus infection. The method will be especially effective with respect to the treatment or prevention of sexually transmitted diseases and viruses that infect the eye. Examples of specific viruses that are amenable to this approach include: herpes simplex virus type 1; herpes simplex virus type 2; human papillomavirus; human immunodeficiency virus; and human cytomegalovirus. In the case of sexually transmitted viruses (e.g., human immunodeficiency virus or human papillomavirus), RNA may be delivered by means of a contraceptive device. In the case of an eye infection, the RNA may be delivered using a solution in the form of eye drops. In addition, RNA may be directly injected into benign or malignant tumors to induce cellular apoptosis. In general, it is expected that 0.1-100 ml of the RNA pharmaceutical composition will be administered per gram of tumor. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A. Making of tiRNAs 
     Methods for chemically synthesizing short strands of RNA are well known in the art (see e.g., Usman, et al.,  J. Am. Chem. Soc.  109:7845 (1987); Scaringe et al.,  Nucl. Ac. Res.  18:5433 (1990); Wincott et al.,  Nucl. Ac. Res.  23:2677 (1995); Wincott et al.,  Methods Mol. Biol.  74:59 (1997) Milligan,  Nucl. Ac. Res.  21:8783 (1987), all of which are hereby incorporated by reference in their entirety) and make use of common nucleic acid protecting and coupling groups. Syntheses may be performed on commercial equipment designed for this purpose, e.g., a 394 Applied Biosystems, Inc. synthesizer, using protocols supplied by the manufacturer. Any of these methods or alternative methods known in the art may be used to make the RNA of the present invention. 
     B. Pharmaceutical Compositions 
     The RNAs may be administered to patients in a pharmaceutical composition comprising the nucleic acids along with a pharmaceutically acceptable carrier or excipient. Carriers may be any solvent, diluent, liquid or solid vehicle that is pharmaceutically acceptable and typically used in formulating drugs. Guidance concerning the making of pharmaceutical formulations can be obtained from standard works in the art (see, e.g.,  Remington&#39;s Pharmaceutical Sciences,  16 th  edition, E. W. Martin, Easton, Pa. (1980)). In addition, pharmaceutical compositions may contain any of the excipients that are commonly used in the art. Examples of carriers or excipients that may be present include, but are not limited to, sugars (e.g., lactose, glucose and sucrose); starches, such as corn starch or potato starch; cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, ethyl cellulose, or cellulose acetate); malt; gelatin; talc; cocoa butter; oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, or soybean oil); glycols; buffering agents; saline; Ringer&#39;s solution; alcohols; lubricants; coloring agents; dispersing agents; coating agents; flavoring agents; preservatives; or antioxidants. 
     C. Route of Delivery 
     The invention is compatible with the delivery of RNAs by any route known in the art, including peroral, intravaginal, internal, rectal, nasal, lingual, transdermal, intravenous, intra-arterial, intramuscular, intraperitoneal, intracutaneous and subcutaneous routes. The most preferred route is either topically or by local injection. It will also be understood that the RNAs may be in any pharmaceutically acceptable form of including pharmaceutically acceptable salts 
     Liquid dosage forms for oral or topical administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, creams, ointments and elixirs. In addition to the active compounds, liquid dosage form may contain inert diluents commonly used in the art, such as, for example, water, or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils, glycerol, alcohols, polyethylene glycols, and fatty acid esters. Injectable preparations may be in the form of sterile, injectable aqueous or oleaginous suspensions. Examples of diluents or solvents that may be used include 1,3-butanediol, water, Ringer&#39;s solution and isotonic saline solutions. In addition, oils or fatty acids may be present. 
     Pharmaceutical compositions may be given to a patient in one or more unit dosage forms. A “unit dosage form” refers to a single drug administration entity, e.g., a single tablet, capsule or injection vial. The amount of RNA present should be at least the amount required to inhibit protein synthesis by 10% and/or induce apoptosis in 10% of cells with higher percentages being preferred. The exact dosages may be determined for individual tiRNAs using methods that are well known in the art of pharmacology and may be further adjusted by physicians on a case-by-case basis based upon clinical considerations. 
     D. Methods for Delivering tiRNAs to Cells 
     Protocols for delivering RNA to cells have been described in many references including: Akhtar, et al.,  Trends Cell Biol.  2:139 (1992); WO 94/02595; WO99/04819; WO93/23569; and WO99/05094. Methods for administered nucleic acids to cells include: encapsulation in liposomes; by iontophoresis; or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. Alternatively, tiRNA may be locally delivered by direct injection or by use of an infusion pump. For a review of drug delivery strategies see Ho et al.,  Curr. Opin. Mol. Ther.  1:336 (1999) and Groothuis, et al.,  J. NeuroVirol.  3:387 (1997)) All of these references are hereby incorporated by reference in their entirety and may be used in conjunction with the present invention. 
     E. Treatment Methods 
     Cells growing in vitro may be contacted by the tiRNAs described herein in order to help in the study of apoptosis. Preparations may also be applied topically to skin lesions, e.g., lesions associated with melanoma. The exact dosage will be determined using procedures well known in the art, balancing toxicity and therapeutic efficacy. Compounds may also be given to test animals to study their effect. In these cases, dosages are limited only by toxicity. It should also be recognized that inhibitory compounds may be administered as the sole active agents in a dosage form, or they may be combined with other drugs to improve overall effectiveness. 
     EXAMPLES 
     The present example demonstrates that mammalian cells subjected to arsenite-induced oxidative stress, heat shock, or UV-irradiation activate a tRNA-anticodon nuclease to produce tRNA-derived, stress-induced RNAs (tiRNAs). Synthetic tiRNAs corresponding to the 5′, but not the 3′, end of tRNA inhibit protein translation in both reticulocyte lysates and transfected cells. The production and activity of tiRNAs is inversely correlated with the phosphorylation of eIF2α, suggesting that tiRNAs are components of a phospho-eIF2α-independent stress response program. 
     A. Experimental Procedures 
     Cell Culture and Treatment 
     U2OS cells were cultured in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (Sigma) and antibiotics (penicillin (100 U/ml) and streptomycin (100 μg/ml). Lipofectamine 2000 (Invitrogen) and Optimem medium (Life Technologies) were used for transfection of tiRNAs and siRNAs. Wild type (SS) and S51A knock-in (AA) mouse embryonic fibroblasts (MEFs), were cultured in DMEM with 10% fetal calf serum and antibiotics. For stress induction, various doses of sodium arsenite (Sigma) were added in medium. The cells were washed with PBS twice before UV irradiation (UV crosslinker FB-UVXL-1000 (FisherBiotech)). Heat shock was achieved by incubating cells in a 42° C. oven. 
     RNA Analysis 
     Total RNA was extracted by using Trizol (Invitrogen). RNA (10 μg per well) was analyzed using TBS-Urea Gels (Invitrogen) or 1.1% agarose/2% formaldehyde MOPS gels, transferred to Nytran Supercharge membranes (Schleicher and Schuell) and hybridized overnight at 50° with digoxigenin-labeled DNA probes in DIG Easy Hyb solution (Roche). After washing at 60° with 2×SSC/0.1% SDS (10 min) and 0.5×SSC/0.1% SDS (20 min, 2 times), the membranes were blocked in Blocking Reagent (Roche) for 30 min at room temperature, probed with alkaline phosphatase-labeled anti-digoxigenin antibody (Roche) for 30 min and washed for 30 min with 130 mM TrisHCl pH 7.5/100 mM NaCl/0.3% Tween-20. Signals were visualized with CDP-Star (Roche). Probes for Httn-Q82, 28S rRNA and 18S rRNA were generated from U20S cDNA by PCR using digoxigenin-labeled nucleotides (Roche) and primer pairs (S198/S199 (Httn-Q82), S217/S218 (28S rRNA) and S215/S216 (18S rRNA), respectively). DIG labeled probes for 5S rRNA, tRNA and tiRNA were prepared by using DIG Oligonucleotide 3′-End Labeling Kit, 2 nd  Generation (Roche) according to the manufacturer&#39;s protocol. 
     In Vitro Translation Assays 
     Flexi Rabbit Reticulocyte Lysate System (Promega) was used for in vitro luciferase translation according to the manufacturer&#39;s protocol. TNT Quick Coupled Transcription/Translation System (Promega) was used for in vitro Httn-Q82 reporter plasmid according to the manufacturer&#39;s protocol. Translation reactions were performed in a total volume of 10 μl at 30° C. for 30 minutes for luciferase and 12.5 μl at 30° C. for 90 minutes for Httn-Q82. The in vitro translated protein product was detected by immunoblot. 
     Biotinylated tiRNA Ala  Pull Down Assay 
     To enrich tiRNA-associated RNPs, biotinylated tiRNA Ala  and a biotinylated control RNA were added to an in vitro translation mixture (Flexi Rabbit Reticulocyte Lysate System) with luciferase mRNA in a total volume of 20 μl at 30° C. for 30 minutes. The reactions were diluted by the addition of binding buffer (20 mM Hepes, pH 7.9, 300 mM NaCl, 10 mM MgCl 2 , 0.3% TritonX) containing RNasin-Plus RNase Inhibitor (Promega), Halt protease inhibitor cocktail and Halt phosphatease inhibitor cocktail (Pierce). The biotinylated RNA bound molecules were captured by Streptavidine Magnetic Particles (Roche) for 30 minutes at room temperature. The particles were washed with 1 ml binding buffer 6 times, then 33% of the particles were re-suspended in 2× sample buffer for western blot and 66% of the particles were extracted using TRIzol for northern blot. 
     Sucrose Gradient Analysis 
     U2OS cells at ˜90% confluence were treated with or without SA (500 μM) for 90 minutes. The conditioned cells were washed in Hanks&#39; balanced salt solution containing 5 μg/ml cycloheximide, and then scrape harvested and centrifuged. Pellets were lysed in 1 ml of ice-cold lysis buffer (300 mM NaCl, 15 mM Tris (pH 7.4), 15 mM MgCl 2 , 1% TritonX, 0.5 U/ml, 5 μg/ml cycloheximide, RNasin-Plus RNase Inhibitor, Halt protease inhibitor cocktail. The cell suspension was incubated at 4° for 10 minutes. The sample was subjected to microfuge centrifugation for 15 min at 14,000 rpm. The resulting supernatant was then layered onto preformed 10-50% linear sucrose gradients (made up in 300 mM NaCl, 15 mM Tris (pH 7.4), 15 mM MgCl 2 , 5 μg/ml cycloheximide, 14 mM 2-mercaptoethanole) over a 60%-0.5-ml sucrose cushion in 11 ml tubes (Beckman). Centrifugation was performed at 35,000 rpm for 190 minutes (for polysome fraction collection) or 250 minutes (for monosome fraction collection) using a Beckman SW40Ti rotor. Gradients were eluted from the top using a Brandel elution system (Brandel, Gaithersburg, Md.). The eluate was continuously monitored at 254 nm using an ISCO UA5 UV monitor (ISCO, Lincoln, Nebr.). Fractions were collected from the top of the gradient. Total RNA was extracted from individual fractions, and 0.5 μg of RNA was resolved by TBS-Urea Gel (Invitrogen) or 1.1% agarose/2% formaldehyde MOPS gel for Northern blotting. 
     Metabolic Labeling 
     Control RNA, 5′ or 3′ tiRNA Ala  were transfected into U2OS cells in 24 well plates and cultured for various times. The cells were incubated with labeling medium (D-MEM without L-glutamine, sodium pyruvate, L-methionine or L-cystine, Invitrogen (Invitrogen)) supplemented with 5% dialyzed Fetal Bovine Serum (HyClone)) for 30 minutes, replaced with fresh labeling medium containing 150˜250 μCi of L- 35 S methionine/well (EasyTag™ EXPRESS35S Protein Labeling Mix) and incubated for 30 minutes. After washing with PBS twice, cells were harvested in 400 μl lysis buffer (2% SDS/20 mM Hepes, pH=7.4), sonicated, and the protein was precipitated by addition of 60% acetone. The proteins were re-suspended in lysis buffer and 10 μl of each sample in Ecoscint H (National Diagnostics) was counted using a liquid scintillation counter (Beckman, LS5801). Protein concentration was determined by Protein Assay BCA Protein Assay kit (PIERCE). 
     TUNEL Staining 
     U2OS cells grown on coverslips were transfected with control RNA, 5′ or 3′ tiRNA Ala  for 12 hours using lipofectamine. TUNEL assay was performed by using ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (Chemicon) according to the manufacture&#39;s instruction followed by counter staining with Hoechst 33258. Cells were visualized using a Nikon Eclipse 800 microscope, and images were digitally captured using a CCD-SPOT RT digital camera and compiled using Adobe® Photoshop® software (v6.0). 
     B. Results and Discussion 
     Extracts prepared from human U2OS cells exposed to arsenite-induced oxidative stress, heat shock, or UV-irradiation were separated on a denaturing gel and developed with SYBR Gold to visualize stress-induced small RNAs. Northern blotting using cDNA probes complementary to the 5′ end of tRNA Met  and the 5′ and 3′ ends of various tRNAs revealed that these stress-induced RNAs are produced by tRNA cleavage. The size of these fragments requires that cleavage occur, as in Tetrahymena, in or near the anticodon loop. tiRNAs are rapidly induced (within 20 minutes) in response to arsenite-mediated oxidative stress and persist for at least 11 hours in cells allowed to recover from stress. The phosphorylation and dephosphorylation of eIF2α over this time course provided a marker of stress and recovery from stress. Arsenite-induced tiRNAs are observed in several different primate cell lines, indicating that this phenomenon is widespread in mammalian cells. 
     To determine the potential for tiRNAs to mediate phospho-eIF2α-independent translational arrest, we compared their induction in mouse embryo fibroblasts (MEFs) derived from wild type or eIF2α (S51A) mutant mice (Scheuner, et al.,  Mol Cell  7:1165-1176 (2001)). The expression of mature tRNA Met  is similar in wild type (wt) and mutant (mut) cells in the absence or presence of arsenite (SA). In contrast, the induction of tiRNA Met  is significantly greater in mutant cells, compared to wild type cells, indicating that phospho-eIF2α is not required for, and may inhibit, tiRNA production. This conclusion is supported by an enhanced production of tiRNAs in U2OS cells treated with control or heme-regulated initiation factor 2-α kinase (HRI)-specific siRNAs. Knock down of HRI, the eIF2α kinase activated by arsenite, increases the arsenite-induced production of tiRNAs. Taken together, these results indicate that stress-induced induction of tiRNAs does not require phospho-eIF2α. Moreover, phospho-eIF2α appears to suppress the induction of tiRNA. 
     We hypothesized that tiRNAs may inhibit translation by interfering with some aspect of tRNA function. Endogenous tiRNAs from arsenite-treated U2OS cells were gel-purified to enrich for small RNA populations including 5′ and 3′ tRNA fragments. These heterogeneous populations of small RNA were found to modestly inhibit the translation of luceriferase transcripts in reticulocyte lysates. To determine whether small RNAs corresponding to specific 5′ or 3′ tRNA fragments also suppress protein translation, we added synthetic tiRNAs (sequences corresponding to piwi-associated tRNA fragments) to reticulocyte lysates and quantified the synthesis of the luciferase reporter protein. Synthetic 5′ tiRNA Ala , but not 3′ tiRNA Ala , was found to inhibit protein translation in a dose dependent manner. The potency of 5′ tiRNAs derived from different tRNAs differs reproducibly, with a rank order: 5′ tiRNA Ala &gt;5′ tiRNA Pro &gt;5′ tiRNA Gly &gt;5′ tiRNA Gln . In a mixed transcription/translation system, synthetic 5′ tiRNAs corresponding to gln, val, and met tRNAs were found to similarly inhibit the production of huntingtin protein without affecting huntingtin mRNA. The ability of 5′ tiRNA Val  to inhibit the translation of huntingtin, a protein that lacks valine residues, reveals that translational repression is codon independent. 
     Separation of extracts from U2OS cells cultured in the absence (−) or presence (SA) of arsenite over sucrose gradients showed that tiRNAs migrate near the top of the gradient and are found in fractions containing 40S, but not 60S ribosomal subunits. To determine whether tiRNA Ala  can bind 40S ribosomal subunits, we compared the ability of biotinylated 5′ tiRNA Ala  and a biotinylated stem loop control RNA to pull down 18S ribosomal RNA from reticulocyte lysates. It was found that biotinylated 5′tiRNA Ala , but not biotinylated stem loop control RNA, pulls down 18S rRNA, consistent with a specific interaction with the small ribosomal subunit. 
     Transfection of synthetic 5′, but not 3′, tiRNA Ala  into U2OS cells induces a dose- and time-dependent inhibition of global protein synthesis. Moreover, synthetic 5′, but not 3′, tiRNA Ala  inhibits global protein synthesis in both wild type and S51A mutant MEFs, indicating that inhibition of protein synthesis does not require phosphorylation of eIF2α. In both U20S cells and MEFs, transfection of 5′, but not 3′, tiRNA Ala  induces obvious toxicity (i.e., rounding up and blebbing) after approximately 9 hours. TUNEL staining showed that U2OS cells accumulate DNA strand breaks consistent with the onset of apoptotic cell death. Thus, both 5′ tiRNA Ala  and phospho-eIF2α inhibit protein synthesis and induce apoptosis in human cells. 
     Our results suggest that a stress-activated ribonuclease targets the anticodon loop of tRNAs to produce regulators of protein translation in mammalian cells. It is possible that intact tRNAs with nicked anticodon loops are an active component of this stress pathway. However, the findings that piwi proteins associate with both 5′ and 3′ tiRNAs, together with the ability of synthetic 5′, but not 3′, tiRNAs to inhibit protein translation and induce apoptosis, supports a role for processed tRNA fragments in this pathway. 
     Stress-induced phosphorylation of eIF2α inhibits translation initiation and triggers apoptotic cell death (Srivastava, et al.,  J. Biol. Chem.  273:2416-2423 (1998)). In viruses that replicate via dsRNA intermediates, PKR-induced phosphorylation of eIF2α triggers global inhibition of protein synthesis. These viruses counter the PKR/eIF2α translation control pathway by inactivating PKR or activating an eIF2α phosphatase (Garcia, et al.,  Biochimie  89:799-811 (2007)). The results described above suggest that stress-induced tRNA cleavage may provide a phospho-eIF2α independent pathway that inhibits protein synthesis and induces apoptosis. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 5′ PiRNA Sequences 
               
             
          
           
               
                 tiRNA 
                   
                   
                   
               
               
                 Homology 
                 PiRNA Sequence 
                 SEQ ID NO. 
                 Reference 
               
               
                   
               
               
                 Ala tRNA 
                 ggggguguagcucagugguagagcgcgugcuu 
                 SEQ ID NO: 1 
                   Homo sapiens  piRNA piR-36256 
               
               
                   
                 ggggguguagcucagugguagagcgcgugcu 
                 SEQ ID NO: 2 
                   Homo sapiens  piRNA piR-36255 
               
               
                   
                 gggggguguagcucagugguagagcgcgugcu 
                 SEQ ID NO: 3 
                   Homo sapiens  piRNA piR-36243 
               
               
                   
                 ggggguguagcucagugguagagcgcgugc 
                 SEQ ID NO: 4 
                   Homo sapiens  piRNA piR-36254 
               
               
                   
                 gggggguguagcucagugguagagcgcgugc 
                 SEQ ID NO: 5 
                   Homo sapiens  piRNA piR-36242 
               
               
                   
                 gggggunuagcucagugguagagcgcgugcuu 
                 SEQ ID NO: 6 
                   Homo sapiens  piRNA piR-36258 
               
               
                   
                 ggggguguagcucagugguagagcgcgug 
                 SEQ ID NO: 7 
                   Homo sapiens  piRNA piR-36253 
               
               
                   
                 guguagcucagugguagagcgcgugcuucgc 
                 SEQ ID NO: 8 
                   Homo sapiens  piRNA piR-36685 
               
               
                   
                 ggggunuagcucagugguagagcgcgugcuu 
                 SEQ ID NO: 9 
                   Homo sapiens  piRNA piR-36272 
               
               
                   
                 ggggggunuagcucagugguagagcgcgugcu 
                 SEQ ID NO: 10 
                   Homo sapiens  piRNA piR-36244 
               
               
                   
                 gggguguagcucagugguagagagcgugcuu 
                 SEQ ID NO: 11 
                   Homo sapiens  piRNA piR-36270 
               
               
                   
                 gggggunuagcucagugguagagcgcgugc 
                 SEQ ID NO: 12 
                   Homo sapiens  piRNA piR-36257 
               
               
                   
                 ggggguguagcucagugguagagagcgugcu 
                 SEQ ID NO: 13 
                   Homo sapiens  piRNA piR-36252 
               
               
                   
                 ggggauguagcucagugguagagcgcaugcu 
                 SEQ ID NO: 14 
                   Homo sapiens  piRNA piR-36225 
               
               
                   
                 gggggauuagcucaaaugguagagcgcucg 
                 SEQ ID NO: 15 
                   Homo sapiens  piRNA piR-36241 
               
               
                   
                 ggggaunuagcucagugguagagcgcaugcu 
                 SEQ ID NO: 16 
                   Homo sapiens  piRNA piR-36229 
               
               
                   
               
               
                 Arg tRNA 
                 ggcucuguugcgcaauggauagcgcau 
                 SEQ ID NO: 17 
                   Homo sapiens  piRNA piR-36082 
               
               
                   
               
               
                 Asp tRNA 
                 uccucauuaguauagugguga guauccc 
                 SEQ ID NO: 18 
                   Homo sapiens  piRNA piR-44312 
               
               
                   
               
               
                 Cys tRNA 
                 ggggguauagcucagugguagagcauuuga 
                 SEQ ID NO: 19 
                   Homo sapiens  piRNA piR-36249 
               
               
                   
                 ggggguauagcucagugguagagcauuug 
                 SEQ ID NO: 20 
                   Homo sapiens  piRNA piR-36248 
               
               
                   
                 ggggguauagcucagugguagagcauuu 
                 SEQ ID NO: 21 
                   Homo sapiens  piRNA piR-36247 
               
               
                   
                 ggggguauagcucaguggguagagcau 
                 SEQ ID NO: 22 
                   Homo sapiens  piRNA piR-36246 
               
               
                   
                 ggggguguaacucagugguagagcauuuga 
                 SEQ ID NO: 23 
                   Homo sapiens  piRNA piR-36251 
               
               
                   
               
               
                 Gln tRNA 
                 gguuccaugguguaaugguuagcacucug 
                 SEQ ID NO: 24 
                   Homo sapiens  piRNA piR-36378 
               
               
                   
               
               
                 Gly tRNA 
                 uuggugguucagugguagaauucucgccugcc 
                 SEQ ID NO: 25 
                   Homo sapiens  piRNA piR-61648 
               
               
                   
                 uuggugguucagugguagaauucucgccugc 
                 SEQ ID NO: 26 
                   Homo sapiens  piRNA piR-61647 
               
               
                   
                 uuggugguucagugguagaauucucgccug 
                 SEQ ID NO: 27 
                   Homo sapiens  piRNA piR-61646 
               
               
                   
                 uggugguucagugguagaauucucgccug 
                 SEQ ID NO: 28 
                   Homo sapiens  piRNA piR-57498 
               
               
                   
                 auuggugguucagugguagaauucucgccug 
                 SEQ ID NO: 29 
                   Homo sapiens  piRNA piR-31925 
               
               
                   
                 uuggugguucagugguagaauucucgccu 
                 SEQ ID NO: 30 
                   Homo sapiens  piRNA piR-61645 
               
               
                   
                 ggcauuggugguucagugguagaauucucgc 
                 SEQ ID NO: 31 
                   Homo sapiens  piRNA piR-35982 
               
               
                   
                 auuggugguucagugguagaauucucgcc 
                 SEQ ID NO: 32 
                   Homo sapiens  piRNA piR-31924 
               
               
                   
                 agcauuggugguucagugguagaauucucgc 
                 SEQ ID NO: 33 
                   Homo sapiens  piRNA piR-31068 
               
               
                   
                 cauuggugguucagugguagaauucucgc 
                 SEQ ID NO: 34 
                   Homo sapiens  piRNA piR-32679 
               
               
                   
                 gggaggcccggguucguuucccggccaaugca 
                 SEQ ID NO: 35 
                   Homo sapiens  piRNA piR-36173 
               
               
                   
                 gcauuggugguucagugguagaauucucac 
                 SEQ ID NO: 36 
                   Homo sapiens  piRNA piR-35284 
               
               
                   
                 cgggaggcccggguucgguucccggccaaugc 
                 SEQ ID NO: 37 
                   Homo sapiens  piRNA piR-33486 
               
               
                   
                 uuggugguucagugguagaauucucgc 
                 SEQ ID NO: 38 
                   Homo sapiens  piRNA piR-61644 
               
               
                   
                 gacauuggugguucagugguagaauucu 
                 SEQ ID NO: 39 
                   Homo sapiens  piRNA piR-34358 
               
               
                   
                 ugguucagugguagaauucucgccucc 
                 SEQ ID NO: 40 
                   Homo sapiens  piRNA piR-57660 
               
               
                   
                 gcauugguauagugguaucaugcaaga 
                 SEQ ID NO: 41 
                   Homo sapiens  piRNA piR-35280 
               
               
                   
                 agcguuggugguauaguggugagcauagcugc 
                 SEQ ID NO: 42 
                   Homo sapiens  piRNA piR-31143 
               
               
                   
               
               
                 His tRNA 
                 ggccgugaucguauagugguuaguacucug 
                 SEQ ID NO: 43 
                   Homo sapiens  piRNA piR-36041 
               
               
                   
                 ucgccgugaucguauagugguuaguacucug 
                 SEQ ID NO: 44 
                   Homo sapiens  piRNA piR-44984 
               
               
                   
                 ggccgugaucguauagugguuaguacuc 
                 SEQ ID NO: 45 
                   Homo sapiens  piRNA piR-36040 
               
               
                   
                 aggccgugaucguauagugguuaguacuc 
                 SEQ ID NO: 46 
                   Homo sapiens  piRNA piR-31355 
               
               
                   
                 ggccgugaucguauagugguuaguacu 
                 SEQ ID NO: 47 
                   Homo sapiens  piRNA piR-36039 
               
               
                   
                 ggccgugaucguauagugguua guac 
                 SEQ ID NO: 48 
                   Homo sapiens  piRNA piR-36038 
               
               
                   
               
               
                 Ile tRNA 
                 ggccgguuagcucaguugguuagagc 
                 SEQ ID NO: 49 
                   Homo sapiens  piRNA piR-36037 
               
               
                   
                 ggccgguuagcucaguuggucagagc 
                 SEQ ID NO: 50 
                   Homo sapiens  piRNA piR-36036 
               
               
                   
                 ggccgguuagcucaguugguaagagcuuggu 
                 SEQ ID NO: 51 
                   Homo sapiens  piRNA piR-36035 
               
               
                   
                 ggggcggccgguuagcucaguugguaagagc 
                 SEQ ID NO: 52 
                   Homo sapiens  piRNA piR-36235 
               
               
                   
                 ggccgguuagcucaguugguaagagc 
                 SEQ ID NO: 53 
                   Homo sapiens  piRNA piR-36034 
               
               
                   
               
               
                 Leu tRNA 
                 gguaguguggccgagcggucuaaggc 
                 SEQ ID NO: 54 
                   Homo sapiens  piRNA piR-36318 
               
               
                   
                 guagucguggccgagugguuaaggcuaugga 
                 SEQ ID NO: 55 
                   Homo sapiens  piRNA piR-36441 
               
               
                   
                 gacgagguggccgagugguuaaggcuauggau 
                 SEQ ID NO: 56 
                   Homo sapiens  piRNA piR-34444 
               
               
                   
                 gacgagguggccgagugguuaaggcuauggac 
                 SEQ ID NO: 57 
                   Homo sapiens  piRNA piR-34443 
               
               
                   
                 gacgagguggccgagugguuaaggcuaugga 
                 SEQ ID NO: 58 
                   Homo sapiens  piRNA piR-34442 
               
               
                   
                 gacgagguggccgagugguuaaggcuaugg 
                 SEQ ID NO: 59 
                   Homo sapiens  piRNA piR-34441 
               
               
                   
                 gacgagguggccgagugguuaaggcaaugga 
                 SEQ ID NO: 60 
                   Homo sapiens  piRNA piR-34440 
               
               
                   
                 gacgagguggccgagugguuaaggcaaugg 
                 SEQ ID NO: 61 
                   Homo sapiens  piRNA piR-34439 
               
               
                   
                 uguagucguggccgagu gguuaaggc 
                 SEQ ID NO: 62 
                   Homo sapiens  piRNA piR-57942 
               
               
                   
               
               
                 Lys tRNA 
                 gccuggauagcucaguugguagagcaucaga 
                 SEQ ID NO: 63 
                   Homo sapiens  piRNA piR-35463 
               
               
                   
                 gccuggauagcucaguugguagagcauca 
                 SEQ ID NO: 64 
                   Homo sapiens  piRNA piR-35462 
               
               
                   
                 gccuggguagcucagucgguagagcaucagac 
                 SEQ ID NO: 65 
                   Homo sapiens  piRNA piR-35469 
               
               
                   
                 gccuggguagcucagucgguagagcaucaga 
                 SEQ ID NO: 66 
                   Homo sapiens  piRNA piR-35468 
               
               
                   
                 gccuggguagcucagucgguagagcaucag 
                 SEQ ID NO: 67 
                   Homo sapiens  piRNA piR-35467 
               
               
                   
               
               
                 Met tRNA 
                 gcagaguggcgcagcggaagcgugcugggccc 
                 SEQ ID NO: 68 
                   Homo sapiens  piRNA piR-35176 
               
               
                   
                 ggcagaguggcgcagcggaagcgugcugggcc 
                 SEQ ID NO: 69 
                   Homo sapiens  piRNA piR-35952 
               
               
                   
                 gcagaguggcgcagcggaagcgugcugg 
                 SEQ ID NO: 70 
                   Homo sapiens  piRNA piR-35175 
               
               
                   
                 ugcagaguggcgcagcggaagcgugcugg 
                 SEQ ID NO: 71 
                   Homo sapiens  piRNA piR-50725 
               
               
                   
                 gcaguggcgcagcggaagcgugcugggcc 
                 SEQ ID NO: 72 
                   Homo sapiens  piRNA piR-35229 
               
               
                   
                 gcagaguggcgcagcggaagcgugcug 
                 SEQ ID NO: 73 
                   Homo sapiens  piRNA piR-35174 
               
               
                   
                 cgcagagucgcgcagcggaagcgugcugggcc 
                 SEQ ID NO: 74 
                   Homo sapiens  piRNA piR-33387 
               
               
                   
                 cagagucgcgcagcggaagcgugcugggccc 
                 SEQ ID NO: 75 
                   Homo sapiens  piRNA piR-32374 
               
               
                   
                 agaguugcgcagcggaagcgugcugggccca 
                 SEQ ID NO: 76 
                   Homo sapiens  piRNA piR-30961 
               
               
                   
                 gagauagcagaguggcgcagcggaagc 
                 SEQ ID NO: 77 
                   Homo sapiens  piRNA piR-30926 
               
               
                   
               
               
                 Pro tRNA 
                 ggcucguuggucuagggguaugauucucgg 
                 SEQ ID NO: 78 
                   Homo sapiens  piRNA piR-36074 
               
               
                   
                 aggcucguuggucuagugguaugauucucg 
                 SEQ ID NO: 79 
                   Homo sapiens  piRNA piR-31368 
               
               
                   
               
               
                 SeC tRNA 
                 gcccggaugauccucaguggucuggggugc 
                 SEQ ID NO: 80 
                   Homo sapiens  piRNA piR-35407 
               
               
                   
               
               
                 Ser tRNA 
                 uguagucguggccgagugguuaaggc 
                 SEQ ID NO: 81 
                   Homo sapiens  piRNA piR-57942 
               
               
                   
                 gacgagguggccgagugguuaaggcuauggac 
                 SEQ ID NO: 82 
                   Homo sapiens  piRNA piR-34443 
               
               
                   
                 gacgagguggccgagugguuaaggcuauggau 
                 SEQ ID NO: 83 
                   Homo sapiens  piRNA piR-34444 
               
               
                   
                 gacgagguggccgagugguuaaggcuaugga 
                 SEQ ID NO: 84 
                   Homo sapiens  piRNA piR-34442 
               
               
                   
                 gacgagguggccgagugguuaaggcaaugga 
                 SEQ ID NO: 85 
                   Homo sapiens  piRNA piR-34440 
               
               
                   
                 gacgagguggccgagugguuaaggcuaugg 
                 SEQ ID NO: 86 
                   Homo sapiens  piRNA piR-34441 
               
               
                   
                 gacgagguggccgagugguuaaggcaaugg 
                 SEQ ID NO: 87 
                   Homo sapiens  piRNA piR-34439 
               
               
                   
               
               
                 Sup tRNA 
                 gccuggauagcucaguugguagagcaucaga 
                 SEQ ID NO: 88 
                   Homo sapiens  piRNA piR-35463 
               
               
                   
                 gccuggauagcucaguugguagagcauca 
                 SEQ ID NO: 89 
                   Homo sapiens  piRNA piR-35462 
               
               
                   
               
               
                 Thr tRNA 
                 ggcagaguggcgcagcggaagcgugcugggcc 
                 SEQ ID NO: 90 
                   Homo sapiens  piRNA piR-35952 
               
               
                   
                 gcagaguggcgcagcggaagcgugcugggccc 
                 SEQ ID NO: 91 
                   Homo sapiens  piRNA piR-35176 
               
               
                   
                 gcagaguggcgcagcggaagcgugcugg 
                 SEQ ID NO: 92 
                   Homo sapiens  piRNA piR-35175 
               
               
                   
                 cggaagcgugcugggcccauaacccaga 
                 SEQ ID NO: 93 
                   Homo sapiens  piRNA piR-33437 
               
               
                   
                 ugcagaguggcgcagcggaagcgugcugg 
                 SEQ ID NO: 94 
                   Homo sapiens  piRNA piR-50725 
               
               
                   
                 gcaguggcgcagcggaagcgugcugggcc 
                 SEQ ID NO: 95 
                   Homo sapiens  piRNA piR-35229 
               
               
                   
                 gcagaguggcgcagcggaagcgugcug 
                 SEQ 1D NO: 96 
                   Homo sapiens  piRNA piR-35174 
               
               
                   
                 cgcagagucgcgcagcggaagcgugcugggcc 
                 SEQ ID NO: 97 
                   Homo sapiens  piRNA piR-33387 
               
               
                   
                 cagagucgcgcagcggaagcgugcugggccc 
                 SEQ ID NO: 98 
                   Homo sapiens  piRNA piR-32374 
               
               
                   
                 agaguugcgcagcggaagcgugcugggccca 
                 SEQ ID NO: 99 
                   Homo sapiens  piRNA piR-30961 
               
               
                   
               
               
                 Tyr tRNA 
                 gccuggauagcucaguugguagagcaucaga 
                 SEQ ID NO: 100 
                   Homo sapiens  piRNA piR-35463 
               
               
                   
                 gccuggauagcucaguugguagagcauca 
                 SEQ ID NO: 101 
                   Homo sapiens  piRNA piR-35462 
               
               
                   
               
               
                 Val tRNA 
                 uuccguaguguagugguuaucacguucgccuc 
                 SEQ ID NO: 102 
                   Homo sapiens  piRNA piR-60577 
               
               
                   
                 uuccguaguguagugguuaucacguucgcc 
                 SEQ ID NO: 103 
                   Homo sapiens  piRNA piR-60576 
               
               
                   
                 uccguaguguagugguuaucacguucgccuga 
                 SEQ ID NO: 104 
                   Homo sapiens  piRNA piR-43996 
               
               
                   
                 uccguaguguagugguuaucacguucgccug 
                 SEQ ID NO: 105 
                   Homo sapiens  piRNA piR-43995 
               
               
                   
                 uccguaguguagugguuaucacguucgccuca 
                 SEQ ID NO: 106 
                   Homo sapiens  piRNA piR-43994 
               
               
                   
                 uccguaguguagugguuaucacguucgccu 
                 SEQ ID NO: 107 
                   Homo sapiens  piRNA piR-43993 
               
               
                   
                 guuuccguaguguaguggucaucacguucgcc 
                 SEQ ID NO: 108 
                   Homo sapiens  piRNA piR-36743 
               
               
                   
                 ccguaguguagugguuaucacguucgcc 
                 SEQ ID NO: 109 
                   Homo sapiens  piRNA piR-33164 
               
               
                   
                 guuuccguaguguaguggucaucacguucgc 
                 SEQ ID NO: 110 
                   Homo sapiens  piRNA piR-36742 
               
               
                   
                 cguaguguagugguuaucacguucgcc 
                 SEQ ID NO: 111 
                   Homo sapiens  piRNA piR-33520 
               
               
                   
                 uccguaguguagugguuaucacuuucgccu 
                 SEQ ID NO: 112 
                   Homo sapiens  piRNA piR-43997 
               
               
                   
                 uccguaguguacugguuaucacguucgccug 
                 SEQ ID NO: 113 
                   Homo sapiens  piRNA piR-43992 
               
               
                   
                 cguaguguaguggucaucacguucgccu 
                 SEQ ID NO: 114 
                   Homo sapiens  piRNA piR-33519 
               
               
                   
                 ggggguguagcucagugguagagcgcgugcuu 
                 SEQ ID NO: 115 
                   Homo sapiens  piRNA piR-36256 
               
               
                   
                 ggggguguagcucagugguagagcgcgugcu 
                 SEQ ID NO: 116 
                   Homo sapiens  piRNA piR-36255 
               
               
                   
                 ggggguguagcucagugguagagcgcgugc 
                 SEQ ID NO: 117 
                   Homo sapiens  piRNA piR-36254 
               
               
                   
                 ggggguguagcucagugguagagcgcgug 
                 SEQ ID NO: 118 
                   Homo sapiens  piRNA piR-36253 
               
               
                   
                 gggggguguagcucagugguagagcgcgugcu 
                 SEQ ID NO: 119 
                   Homo sapiens  piRNA piR-36243 
               
               
                   
                 uuggugguucagugguagaauucucgccugcc 
                 SEQ ID NO: 120 
                   Homo sapiens  piRNA piR-61648 
               
               
                   
                 uuggugguucagugguagaauucucgccugc 
                 SEQ ID NO: 121 
                   Homo sapiens  piRNA piR-61647 
               
               
                   
                 uuggugguucagugguagaauucucgccug 
                 SEQ ID NO: 122 
                   Homo sapiens  piRNA piR-61646 
               
               
                   
                 uuggugguucagugguagaauucucgccu 
                 SEQ ID NO: 123 
                   Homo sapiens  piRNA piR-61645 
               
               
                   
                 uggugguucagugguagaauucucgccug 
                 SEQ ID NO: 124 
                   Homo sapiens  piRNA piR-57498 
               
               
                   
                 auuggugguucagugguagaauucucgccug 
                 SEQ ID NO: 125 
                   Homo sapiens  piRNA piR-31925 
               
               
                   
                 auuggugguucagugguagaauucucgcc 
                 SEQ ID NO: 126 
                   Homo sapiens  piRNA piR-31924 
               
               
                   
                 ggcauuggugguucagugguagaauucucgc 
                 SEQ ID NO: 127 
                   Homo sapiens  piRNA piR-35982 
               
               
                   
                 uuggugguucagugguagaauucucgc 
                 SEQ ID NO: 128 
                   Homo sapiens  piRNA piR-61644 
               
               
                   
                 ugguucagugguagaauucucgccucc 
                 SEQ ID NO: 129 
                   Homo sapiens  piRNA piR-57660 
               
               
                   
                 cauuggugguucagugguagaauucucgc 
                 SEQ ID NO: 130 
                   Homo sapiens  piRNA piR-32679 
               
               
                   
                 agcauuggugguucagugguagaauucucgc 
                 SEQ ID NO: 131 
                   Homo sapiens  piRNA piR-31068 
               
               
                   
                 gcauuggugguucagugguagaauucucac 
                 SEQ ID NO: 132 
                   Homo sapiens  piRNA piR-35284 
               
               
                   
               
             
          
         
       
     
     All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by those of skill in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.