in the last two decades it has become increasingly clear that a diverse range of rnas play numerous critical roles in the regulation of gene expression ( 13 ) . it has also become apparent that rnas hardly function alone in a cellular environment . prominent examples include the pre - mrna splicing machinery , the apparatus that assembles ribosomes and mrna storage particles ( 58 ) . a wealth of data indicate that structure and composition of such ribonucleoprotein complexes ( rnps ) are not static , and that changes in rnp structure and/or composition need to be accurately timed to ensure correct rnp function ( 911 ) . one group of enzymes pivotal for facilitating rearrangments of cellular rnps are the dexh / d proteins ( 11,12 ) . these enzymes are known to manipulate rna structure in an atp - dependent manner ( 13 ) . members of the dexh / d protein family are present in all forms of cellular life and also in several viruses ( 12,14 ) . dexh / d proteins are highly conserved , sharing at least eight characteristic sequence motifs ( 12 ) . the characteristic sequence motif ii often takes the form dead , deah or dexh [ in single letter amino acid code ( 15 ) ] . these signatures provide the names for the three subgroups of the protein family ( 12 ) . the name dexh / d proteins stems from  averaging the signatures of the three subgroups . dexh / d proteins are involved in virtually all aspects of rna metabolism ( 12 ) . significant evidence suggests that these proteins act as atp - driven motors or switches at very specific points in processes such as pre - mrna splicing or during ribosome biogenesis ( 12 ) . yet , it is unknown where precisely the vast majority of dexh / d proteins bind to their targets and which exact conformational changes these proteins catalyze in their respective substrates . nonetheless , physiological functions of many dexh / d proteins correlate with the ability of the enzymes to hydrolyze atp in an rna - stimulated manner , and/or to unwind rna duplexes in vitro in an atp - dependent fashion ( 16 ) . for these reasons , and because of the intuitive connection between the unwinding of rna secondary structure and conformational changes in rnps , it has long been assumed that rna helicase activity is central to the biological function of dexh / d proteins . in fact , dexh / d proteins are frequently referred to as rna helicases . however , because cellular rnas are invariably complexed with proteins , dexh / d proteins are most likely to encounter rna  protein complexes , rather than the pure rna duplexes that are commonly used to measure rna helicase activity in vitro ( 4,16,17 ) . yet , we are only beginning to understand how dexh / d proteins remodel rnps . here we review recent results indicating that dexh / d proteins can directly target rna  protein interactions both in vivo and in vitro and that rnp remodeling does not necessarily require rna duplex unwinding . based on the available data , we outline a basic mechanism by which dexh / d proteins may displace proteins from rna . the first specific examples of dexh / d proteins targeting rna  protein interactions emerged for prp28p , sub2p , prp5p and dbp5p ( 1821 ) . dbp5p is involved in mrna export ( 22 ) , the other three proteins are essential components of the pre - mrna splicing machinery ( 17 ) . the dead - box protein prp28p is involved in exchanging u1 snrna with u6 snrna on the 5 splice site and has been implicated specifically in the removal of the u1 snrnp from the 5 splice site ( 23 ) ( figure 1a ) . one of these stabilizing proteins is u1cp , and prp28 is thought to counteract the stabilizing effect of u1cp ( 18,26 ) . if u1cp contains a mutation that reduces its affinity for the rna , the otherwise essential prp28p becomes dispensable , suggesting that prp28p is responsible for the removal of u1cp ( 18 ) ( figure 1a ) . a similar bypass suppressor strategy illuminated the involvement of the dead - box protein sub2p in the displacement of the protein mud2p ( 19 ) . among other functions , which include a role on rna export ( 27 ) , sub2p participates in early spliceosome assembly by promoting the exchange of the branch point - binding protein ( bbp ) with the u2 snrnp at the pre - mrna branch site ( 28 ) ( figure 1b ) . the binding of bbpp to the branch site is presumably stabilized by the non - essential protein mud2p ( 29 ) . deletion of mud2 obviates the need for the essential sub2p , consistent with a role of sub2p in mud2p displacement ( 19 ) . prp5p is required for the stable addition of the u2 snrnp to the branch site , which normally depends on the atpase activity of prp5p ( 30 ) . however , the interaction of the u2 snrnp with the branch site can also occur with atpase - deficient prp5p , but only if the non - essential protein cus2p is deleted ( 20 ) . thus , the essential prp5p can not be completely bypassed by deletion of cus2p , yet the normally essential atpase activity of prp5p can be made obsolete ( 20 ) , suggesting that prp5p dislodges cus2p in an atp - dependent manner ( figure 1c ) . recently , it has been shown that the dead - box protein dbp5p , which functions in late steps of mrna export on the cytoplasmic side of the nuclear pore complex , is also likely to be specifically required for displacement of the protein mex67 from rna ( 21 ) . it was shown that in dbp5 mutant cells , the mrna export receptor mex67 accumulates on mrna , and that these mex67 bound rnas were enriched at the nuclear rim ( 21 ) . the accumulation of mex67 bound rnas in dbp5 mutant cells were suppressed by a mex67 mutation , consistent with a scenario where dbp5p removes mex67 from the rna ( figure 1d ) . although the observations for prp28p , sub2p , prp5p and dbp5p strongly suggested the involvement of these enzymes in the removal of other proteins from rna , the mechanisms by which dexh / d enzymes caused protein displacements remained unclear ( 17 ) . for example , the data obtained with prp28p , sub2p , prp5p and dbp5 did not illuminate whether dexh / d proteins rely on other cofactors or on a specific context to displace proteins , or whether dexh / d helicases alone are sufficient to dislodge other proteins . it also remained unclear whether dexh / d proteins are able to act directly on the respective rna protein interaction , or whether the enzymes displace proteins only indirectly , possibly through the remodeling of rna secondary structure . to elucidate the basic mechanism(s ) of protein displacement by dexh / d proteins , it is critical to quantitatively analyze rnp remodeling reactions . since it is unknown where precisely the vast majority of dexh / d proteins bind to their targets and which exact conformational changes the enzymes catalyze in their respective substrates ( 12,15 ) , it has not yet been possible to devise model systems that recapitulate a physiological rnp remodeling reaction that can be analyzed quantitatively as well . for example , complex in vitro systems such as pre - mrna splicing extracts provide invaluable qualitative information about dexh / d protein function , but the limited control over parameters such as concentrations of individual factors precludes the use of these systems for quantitative mechanistic studies . thus , it has been only possible to obtain quantitative mechanistic information about rnp remodeling by dexh / d proteins with simple , yet non - physiological rnp models ( 3133 ) . the first rnp used for such model studies consisted of two rna strands which formed a binding site for the prototypical rna - binding protein u1a ( 31 ) ( figure 2a ) . u1a is part of the pre - mrna splicing machinery , and it also acts as a feedback inhibitor for its own gene expression ( 34,35 ) . in the model rnp , u1a forms a homo - dimer on the rna , contacting predominantly the single - stranded loops embedded in the helical regions of the rna ( 36 ) ( figure 2a ) . it was tested whether the dexh rna helicase nph - ii could displace u1a from the rna ( 31 ) . nph - ii , which is involved in the replication of vaccinia virus ( 37 ) , was selected for these protein displacement studies because a basic mechanism existed for the rna unwinding activity of this enzyme ( 38 ) . nph - ii increased the dissociation rate constant of u1a from the rna by more than three orders of magnitude in an atp - dependent fashion ( 31 ) . wait until u1a dissociated spontaneously to then unwind the u1a - binding site ; rather , nph - ii actively displaced u1a ( 31 ) . further kinetic analysis of the rnp remodeling showed that u1a displacement was in fact faster than rna unwinding by nph - ii . the processivity of nph - ii was decreased , but not completely eliminated by the u1a displacement event , i.e. the enzyme could continue to unwind rna duplexes after dislodging u1a without first leaving the rna ( 31 ) . these results established that dexh / d proteins could directly and actively displace stably bound proteins from rna in an atp - dependent reaction . however , observations made with this u1a - based model rnp do not preclude the requirement of other factors or a specific context for protein displacement by other dexh / d proteins . while the above results clearly showed active protein displacement by nph - ii , it remained unclear whether protein displacement required the unwinding of rna secondary structure , which in the u1a - based rnp surrounded the protein - binding site ( figure 2a ) . to test whether dexh / d rna helicases could also displace proteins from rna without unwinding any duplexes , two model rnps were devised where rna secondary structure played no role in protein binding ( 32 ) . one complex was formed between the tryptophan rna - binding attenuation protein ( trap ) and its specific 53-nt - long cognate rna ( figure 2b ) . trap binds to this rna in a sequence - specific manner as a 11-unit oligomer , and its affinity can be modulated by tryptophan ( 39 ) . the second complex was the multi - component exon junction complex ( ejc ) that is deposited on mrnas as a consequence of splicing ( 40 ) ( figure 2c ) . while the exact composition of the ejc is presently unclear , the complex is known to contain at least five distinct proteins that bind tightly in a non - sequence - specific manner 20 nt upstream of exon junctions ( 40 ) . the ejc plays a variety of roles in postprocessing mrna metabolism , including nonsense - mediated decay and translational efficiency ( 41 ) . in an atp - dependent fashion , nph - ii accelerated the dissociation rate constants for both the trap and the ejc - based complexes by several orders of magnitude ( 32 ) . thus , nph - ii actively displaced protein complexes from unstructured rna , indicating that unwinding of rna duplexes was not required for the removal of proteins from rna ( 32 ) . however , while nph - ii actively disrupted both rnps , the ejc was displaced at a significantly slower rate than trap , suggesting that the properties of a given rnp might affect the rate by which it can be remodeled by dexh / d proteins ( 32 ) ( table 1 ) . the ability of a dexh / d protein to perform atp - driven conformational work on single - stranded rna is thought to resemble the movement ( tracking ) of the enzyme on single - stranded nucleic acids that has been observed for some sf1 dna helicases ( 42 ) . a notable difference may be the inability of many dexh / d proteins to move with high processivity on the rna . many dexh / d proteins may only be able to track one or a few steps before dissociating from the rna . the active displacement of u1a , trap and the ejc by nph - ii indicated that dexh / d proteins could efficiently disrupt rna  protein interactions . however , many physiological rnps , such as the spliceosomal complexes that contain the small nuclear rnas , bind to their targets through a combination of rna  rna and rna  protein interactions ( 11 ) . to illuminate the range of rnps which dexh / d proteins can disassemble , it was tested whether nph - ii could displace the u1snrnp , a complex that bound its target through such a combination of rna  rna and multiple rna the u1snrnp is part of the eukaryotic splicing apparatus where it is involved in the recognition of the 5 splice site ( 43 ) . nph - ii accelerated the dissociation of purified u1snrnp from a substrate rna ( with an authentic 5 splice site ) in an atp - dependent fashion , indicating that the enzyme could actively disrupt a more complex rna protein interface ( 33 ) ( figure 2d ) . experiments with four simple model rnps had demonstrated the ability of the dexh / d protein nph - ii to actively displace a diverse range protein complexes from rna ( table 1 ) . it was then critical to test whether the efficient rnp remodeling seen with nph - ii was also observed for other dexh / d proteins , especially for enzymes that displayed lower atpase and rna helicase activties than nph - ii ( 32 ) . therefore , protein displacement was measured for the dead - box protein ded1 from saccharomyces cerevisiae , which is phylogenetically distant from nph - ii ( 32,33 ) . ded1 efficiently unwinds short rna duplexes ( 45 ) , but the enzyme appears significantly less processive than nph - ii ( e. jankowsky , et al . the rna - stimulated atpase activity of ded1 is also considerably lower than that of nph - ii ( 45 ) . nevertheless , ded1 actively displaced the ejc and the u1snrnp from their respective rnas , even with an efficiency comparable to that seen with nph - ii ( table 1 ) . however , ded1 was unable to accelerate the dissociation of u1a or trap from their target rnas ( table 1 ) . in fact , the kinetics for separation of the rna strands of the u1a - based rnp with u1a bound closely matched the kinetics of spontaneous u1a dissociation , indicating that ded1 did not actively displace u1a from the rna ( 33 ) . collectively , these data demonstrated that the ability of dexh / d proteins to actively remodel rnps is not restricted to nph - ii . yet , different enzymes do not necessarily disrupt the same range of rnp substrates in an active fashion . to understand the functional differences between the two helicases , it will be critical to elucidate which features make an rnp susceptible to an active disruption by ded1 . although no results have yet been reported that specifically address this question , the available data suggest that basic macroscopic physicochemical properties of the rnp such as the affinity for the rna and the dissociation rate constant are unlikely to dictate whether a given rnp complex will be actively disassembled by ded1 ( table 1 ) . however , it is worth noting that ded1 actively disrupted the two rnps that bind their target rna through multiple different proteins , whereas the enzyme failed to actively displace the two homo - oligomers u1a and trap . perhaps the arrangement of individual protein units within an rnp may determine whether ded1 can actively disrupt a given rna to reconcile the observations made during the remodeling of the four tested rnps by both nph - ii and ded1 , it is instructive to propose a basic mechanism by which dexh / d proteins actively displace proteins from rna . we emphasize that although the currently available data set is very limited with respect to the diversity of rnps and dexh / d enzymes , a mechanism needs to be consistent with a rather complex set of observations , including ( i ) the ability of both nph - ii and ded1 to displace proteins without unwinding rna secondary structure , ( ii ) the ability of nph - ii to actively displace a greater range of proteins than ded1 , and ( iii ) the capacity of ded1 to actively disassemble the stable multi - component rnps but not the stable homo - oligomeric rnps tested ( table 1 ) . we propose that the ability of a dexh / d protein to actively disrupt a given rnp is based on the capacity of the enzyme to track on single - stranded rna in an atp - dependent fashion , which explains the ability of nph - ii and ded1 to displace proteins without unwinding rna secondary structure . during the tracking , the dexh / d protein is able to capture nucleotides that are normally part of the rna  protein interface of the rnp . this reduction in the number of rna  protein contacts increases the propensity of the protein(s ) to dissociate from the rna ( figure 3a , step 1 ) . additional advance of the enzyme on the rna and the sequestering of more nucleotides further reduces the number of rna  protein contacts in the rnp , eventually leading to the dissociation of the rnp at a rate greater than that of spontaneous rnp dissociation , i.e. the protein is actively displaced from the rna ( figure 3a , step 2 ) . if the dexh / d protein dissociates from the rna before capturing the critical number of nucleotides necessary to accelerate dissociation of the rnp , no active protein displacement is observed ( figure 3a ) . therefore , a dexh / d protein tracking fast and processively ( e.g. nph - ii ) should be able to actively disassemble a wider range of rnps than an enzyme that tracks slowly and/or with low ( or no ) processivity ( e.g. ded1 ) . this prediction is consistent with our observations : the processive nph - ii actively displaces a greater range of proteins than the less processive ded1 . however , active protein displacement is observed even with a less processive enzyme , provided the capture of one ( or few ) nucleotides from the rna for example , a small decrease in the rna  protein interface in a multi - component complex such as the ejc might lead to dissociation of one critical component that in turn unravels the entire rnp ( figure 3b ) . this scenario would provide a basis to explain why ded1 actively disassembles the stable multi - component rnps but not the stable homo - oligomeric rnps tested ( table 1 ) . it is unclear how exactly the nucleotides are captured by the dexh / d proteins . conceivably , the dexh / d helicase could exert force on the other protein to free one or several nucleotides , although it is not known whether dexh / d proteins can produce sufficient force when tracking on rna . alternatively , the dexh / d helicase could simply sequester transiently fraying nucleotides during the tracking on rna . helicase on single - stranded nucleic acid and capture of fraying nucleotides is also considered to be important for the unwinding of dna or rna duplexes by these enzymes ( 46 ) . there , a tracking helicase is proposed to capture fraying nucleotides from the termini of the helix interface ( 46 ) . it is perhaps not surprising that similar mechanisms may underlie both duplex unwinding and protein displacement by because of the limited amount of data for rnp remodeling by dexh / d proteins , other mechanisms by which these enzymes cause active protein displacement should not be discounted . for example , it may be possible that , instead of capturing one or few nucleotides at a time , dexh / d proteins may simply force the entire protein off the nucleic acid by physical clashes between protein domains . while it is unknown whether dexh / d proteins can exert sufficient force when tracking on rna , as mentioned above , it is well established that dexh / d proteins change their domain orientations upon atp binding / hydrolysis ( 47 ) . such large - scale conformational changes could be used to induce a physical clash between the dexh / d enzyme and other proteins . however , this scenario is more difficult to reconcile with the similar rate constants by which ded1 and nph - ii displace the ejc , given the different rates by which both enzymes turn over atp . using simple rnp models to study protein displacement by dexh / d proteins has provided three major insights . first , dexh / d proteins can directly remodel rnps , i.e. the enzymes do not per se require specific cofactors that turn  rna helicases into rnp remodelers . third , not all dexh / d proteins are able to actively disassemble the same range of rnps . the proposed mechanism by which dexh / d proteins actively displace proteins from rna may present a starting point for more targeted questions about the mechanism(s ) of rnp remodeling by dexh / d proteins such as defining conformational changes that dexh / d proteins induce in rna in order to displace proteins . moreover , it will be critical to investigate the rearrangement of more diverse rnps by a larger range of dexh / d proteins . it also remains of central importance to elucidate how dexh / d proteins remodel rnps in vivo . finally , it will be illuminating to compare mechanisms of protein displacement by dexh / d proteins with mechanisms that dna helicases use to remove proteins from dna ( 4850 ) . dexh / d proteins targeting rna  protein interactions : physiological examples . ( a ) displacement of the u1snrnp from an rna containing a 5 splice site by prp28p . the second step shows the atp - independent binding of u2 snrnp to the branch site of the pre - mrna , which is facilitated by prp5 in an atp - independent fashion . dbp5p is bound to the nuclear pore complex on the cytoplasmic side of the nuclear rim . as mrnas are transported through the nuclear pore , dbp5p specifically facilitates the release of mex67 . ovals indicate u1a , lines the rna , and arrows represent the reactions catalyzed by the dexh / d protein . rna removal of the ejc renders the region protected previously from nuclease digestion by the ejc susceptible to degradation . ( d ) removal of the u1 snrnp from a radiolabeled ( asterisk ) rna with a 5 splice site . removal of the u1snrnp was monitored by immunoprecipitation of u1snrnp and subsequent quantification of radioactivity in supernatant and immunoprecipitate . ( a ) schematic representations for the displacement of a single ( homopolymeric ) protein from rna . ( b ) schematic representations for the displacement of a multi - component protein complex from rna . remodeling of four different rnps by ded1 and nph - ii kd , equilibrium binding constant of the protein(complex ) to the rna substrate . koff , dissociation rate constant of the protein ( complex ) from the rna substrate . binding site size judged from the nmr structure of the u1a  rna complex ( 31 ) . in addition , 30 nt in the mrna substrate are protected from digestion with microccocal nuclease ( 33 ) , suggesting an u1snrnp - binding site on the mrna substrate of 30 nt . an equilibrium dissociation constant ( kd ) can not be determined because the ejc is deposited on the rna during pre - mrna splicing ( 32 ) . digestion of the rna with bound ejc using micrococcal nuclease yields two bands of 8 and 10 nt ( 32 ) .
the dexh / d protein family is the largest group of enzymes in eukaryotic rna metabolism . 
 dexh / d proteins are mainly known for their ability to unwind rna duplexes in an atp - dependent fashion . 
 however , it has become clear in recent years that these dexh / d rna helicases are also involved in the atp - dependent remodeling of rna  protein complexes . 
 here we review recent studies that highlight physiological roles of dexh / d proteins in the displacement of proteins from rna . 
 we further discuss work with simple rna 
 protein complexes in vitro , which illuminates mechanisms by which dexh / d proteins remove proteins from rna . 
 although we are only beginning to understand how dexh / d proteins remodel rna  protein complexes , these studies have shown that an 
 rna helicase does not per se require cofactors to displace proteins from rna , that protein displacement does not necessarily involve rna duplex unwinding , and that not all dexh / d proteins are able to disassemble the same range of ribonucleoproteins .