The Long Unwinding Road of RNA Helicases
2007; Elsevier BV; Volume: 27; Issue: 3 Linguagem: Inglês
10.1016/j.molcel.2007.07.014
ISSN1097-4164
AutoresFranziska Bleichert, Susan J. Baserga,
Tópico(s)RNA and protein synthesis mechanisms
ResumoRNA helicases comprise a large family of enzymes that are thought to utilize the energy of NTP binding and hydrolysis to remodel RNA or RNA-protein complexes, resulting in RNA duplex strand separation, displacement of proteins from RNA molecules, or both. These functions of RNA helicases are required for all aspects of cellular RNA metabolism, from bacteria to humans. We provide a brief overview of the functions of RNA helicases and highlight some of the recent key advances that have contributed to our current understanding of their biological function and mechanism of action. RNA helicases comprise a large family of enzymes that are thought to utilize the energy of NTP binding and hydrolysis to remodel RNA or RNA-protein complexes, resulting in RNA duplex strand separation, displacement of proteins from RNA molecules, or both. These functions of RNA helicases are required for all aspects of cellular RNA metabolism, from bacteria to humans. We provide a brief overview of the functions of RNA helicases and highlight some of the recent key advances that have contributed to our current understanding of their biological function and mechanism of action. RNA is probably the most structurally and functionally diverse molecule within living cells. Usually thought of as a messenger of genetic information between DNA and protein (coding RNAs or mRNAs), it also plays a role as an important structural component of macromolecular complexes and can function as a ribozyme to catalyze chemical reactions. Examples of RNA acting as a catalyst include peptide-bond formation, self-splicing, and probably eukaryotic pre-mRNA splicing. Recently, other novel functions of noncoding RNAs have been discovered, including regulation of transcription and translation, RNA stability, heterochromatin formation, and maintenance of genome integrity. The functionality of RNA molecules usually depends on correct folding into the proper tertiary structure and, in ribonucleoprotein complexes (RNPs), also on the association with the correct set of proteins. The function of many small noncoding RNAs involves transient base pairing with a target RNA sequence. Furthermore, many RNPs are dynamic, i.e., they undergo conformational changes that are essential for function and/or they transiently associate with target RNAs through base pairing. All these examples require "helper" proteins such as RNA helicases that can disrupt RNA-RNA or RNA-DNA base pairing, can dissociate proteins from RNA molecules, and assist in proper tertiary structure formation similar to protein chaperones during protein folding. RNA helicases belong to an abundant protein family that is conserved from bacteria to humans. The S. cerevisiae genome, for example, encodes 39 putative helicases that have been implicated in RNA metabolism (SGD and YPD databases; de la Cruz et al., 1999de la Cruz J. Kressler D. Linder P. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families.Trends Biochem. Sci. 1999; 24: 192-198Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). They undoubtedly participate in all aspects of RNA metabolism, ranging from RNA transcription, RNA editing, mRNA splicing, RNA export, rRNA processing, RNA degradation, and RNA 3′ end formation to translation of mRNA into proteins (Table 1). Traditionally, RNA helicases (or unwindases) were defined based on their ability to utilize the energy of NTP binding and hydrolysis to unwind RNA duplexes. However, only in a few cases has the unwinding activity of these helicases been tested and demonstrated in vitro. Recently, it has become clear that the function of RNA helicases is not limited to RNA strand separation. Instead, they can also displace proteins from RNA molecules without duplex unwinding, anneal RNA strands, act as RNA clamps or placeholders, and stabilize on-pathway folding intermediates (Fairman et al., 2004Fairman M.E. Maroney P.A. Wang W. Bowers H.A. Gollnick P. Nilsen T.W. Jankowsky E. Protein displacement by DExH/D "RNA helicases" without duplex unwinding.Science. 2004; 304: 730-734Crossref PubMed Scopus (168) Google Scholar, Halls et al., 2007Halls C. Mohr S. Del Campo M. Yang Q. Jankowsky E. Lambowitz A.M. Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.J. Mol. Biol. 2007; 365: 835-855Crossref PubMed Scopus (100) Google Scholar, Jankowsky et al., 2001Jankowsky E. Gross C.H. Shuman S. Pyle A.M. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase.Science. 2001; 291: 121-125Crossref PubMed Scopus (201) Google Scholar, Shibuya et al., 2004Shibuya T. Tange T.O. Sonenberg N. Moore M.J. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay.Nat. Struct. Mol. Biol. 2004; 11: 346-351Crossref PubMed Scopus (143) Google Scholar, Solem et al., 2006Solem A. Zingler N. Pyle A.M. A DEAD protein that activates intron self-splicing without unwinding RNA.Mol. Cell. 2006; 24: 611-617Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, Yang and Jankowsky, 2005Yang Q. Jankowsky E. ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1.Biochemistry. 2005; 44: 13591-13601Crossref PubMed Scopus (102) Google Scholar). Hence, these proteins have also been called RNA chaperones or RNPases. Despite the multiple functions of this class of proteins, we will continue to refer to them as RNA helicases throughout this review.Table 1 Subfamilies and Established Functions of RNA Helicases in S. cerevisiaeSuperfamilySF2SF1SubfamilyDEADDEAHSki2-likeUpf1-likeRibosome biogenesis Small subunit (SSU)Fal1, Dbp4, Dbp8, Rok1, Rrp3Dhr1, Dhr2 Large subunit (LSU)Dbp2, Dbp3, Dbp6, Dbp7, Dbp9, Dbp10, Drs1, Spb4, Mak5Mtr4 Both subunitsHas1Prp43Sen1mRNA splicingSub2, Prp5, Prp28, Ded1Prp2, Prp16, Prp22, Prp43Brr2RNA exportSub2, Dbp5RNA turnover and quality controlDhh1, Dbp2Ski2, Mtr4Upf1Translation initiationeIF4A,aThe yeast eIF4A is encoded by two different genes: TIF1 and TIF2. Ded1, Dbp1Translation terminationDbp5Upf1, Hel1Translation inhibitionDhh1Ski2, Slh1Small RNA processingMtr4Sen1Mitochondrial RNA metabolismMss116, Mrh4Suv3Data are from the SGD and YPD databases. Please note that some RNA helicases have been proposed to have additional functions in RNA metabolism based on their association with certain large RNPs. Some of those potential functions have been omitted if the functional role was not further verified.a The yeast eIF4A is encoded by two different genes: TIF1 and TIF2. Open table in a new tab Data are from the SGD and YPD databases. Please note that some RNA helicases have been proposed to have additional functions in RNA metabolism based on their association with certain large RNPs. Some of those potential functions have been omitted if the functional role was not further verified. RNA and DNA helicases share conserved helicase motifs, and they provide the basis for classifying helicases into five different superfamilies, designated SF1–5 (Gorbalenya and Koonin, 1993Gorbalenya A.E. Koonin E.V. Helicases: amino acid sequence comparisons and structure-function relationships.Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Google Scholar). Most RNA helicases belong to the SF2 superfamily, and only a few putative RNA helicases, the Upf1-like helicases, belong to SF1 (Table 1). SF1 and SF2 helicases contain seven to nine conserved motifs that constitute the helicase core and are thought to perform similar functions in different proteins. Based on the consensus sequence of the conserved motifs obtained by alignment of helicases within each superfamily, these can be further divided into subgroups: SF2 RNA helicases include the DEAD box, DEAH box, and Ski2-like proteins, generally referred to as DExD/H box RNA helicases, named after one of the consensus amino acid sequence motifs (Caruthers and McKay, 2002Caruthers J.M. McKay D.B. Helicase structure and mechanism.Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (334) Google Scholar, de la Cruz et al., 1999de la Cruz J. Kressler D. Linder P. 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The DEAD box family is by far the largest subgroup within SF2, i.e., 25 out of the 39 putative helicases implicated in RNA metabolism in S. cerevisiae are DEAD box proteins, and most of them are involved in ribosome biogenesis (Table 1). RNA helicases share conserved sequence motifs that are located in two different domains: motifs I, Ia, Ib, II, and III in domain 1 and motifs IV, V, and VI in domain 2 (Figure 1). Based on genetic, biochemical, and structural data, different functions have been assigned to these motifs. The functions of motifs I (Walker A) and II (Walker B) are probably best characterized and are not specific to RNA helicases, as they can also be found in nonhelicase proteins (Walker et al., 1982Walker J.E. Saraste M. Runswick M.J. Gay N.J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.EMBO J. 1982; 1: 945-951Crossref PubMed Google Scholar). Both motifs are required for NTP/ATP binding and catalyze its hydrolysis (Caruthers and McKay, 2002Caruthers J.M. McKay D.B. Helicase structure and mechanism.Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (334) Google Scholar, Cordin et al., 2006Cordin O. Banroques J. Tanner N.K. Linder P. The DEAD-box protein family of RNA helicases.Gene. 2006; 367: 17-37Crossref PubMed Scopus (443) Google Scholar, Rocak and Linder, 2004Rocak S. Linder P. DEAD-box proteins: the driving forces behind RNA metabolism.Nat. Rev. Mol. Cell Biol. 2004; 5: 232-241Crossref PubMed Scopus (383) Google Scholar). Motif III has been suggested to couple ATP binding and hydrolysis to helicase activity, as mutations in this motif drastically interfere with substrate unwinding without affecting ATP binding and hydrolysis in vitro (Pause and Sonenberg, 1992Pause A. Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.EMBO J. 1992; 11: 2643-2654Crossref PubMed Google Scholar, Schwer and Meszaros, 2000Schwer B. Meszaros T. RNA helicase dynamics in pre-mRNA splicing.EMBO J. 2000; 19: 6582-6591Crossref PubMed Google Scholar). This may vary depending on the helicase, as in some helicases mutations in motif III do not support ATP hydrolysis in vitro (Kos and Tollervey, 2005Kos M. Tollervey D. The putative RNA helicase Dbp4p is required for release of the U14 snoRNA from preribosomes in Saccharomyces cerevisiae.Mol. Cell. 2005; 20: 53-64Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Furthermore, the role of this motif in vivo remains somewhat ambiguous, as mutations do not always impair protein function in vivo (Bernstein et al., 2006Bernstein K.A. Granneman S. Lee A.V. Manickam S. Baserga S.J. Comprehensive mutational analysis of yeast DExD/H box RNA helicases involved in large ribosomal subunit biogenesis.Mol. Cell. Biol. 2006; 26: 1195-1208Crossref PubMed Scopus (41) Google Scholar, Granneman et al., 2006aGranneman S. Bernstein K.A. Bleichert F. Baserga S.J. Comprehensive mutational analysis of yeast DExD/H box RNA helicases required for small ribosomal subunit synthesis.Mol. Cell. Biol. 2006; 26: 1183-1194Crossref PubMed Scopus (31) Google Scholar, Hotz and Schwer, 1998Hotz H.R. Schwer B. Mutational analysis of the yeast DEAH-box splicing factor Prp16.Genetics. 1998; 149: 807-815PubMed Google Scholar). Motif VI was shown to function in ATP hydrolysis but had originally also been suggested to have a role in RNA binding (Pause et al., 1993Pause A. Methot N. Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.Mol. Cell. Biol. 1993; 13: 6789-6798Crossref PubMed Scopus (0) Google Scholar). Motifs Ia, Ib, IV, and V are less well studied, but there is evidence that they bind the substrate RNA (Cordin et al., 2006Cordin O. Banroques J. Tanner N.K. Linder P. The DEAD-box protein family of RNA helicases.Gene. 2006; 367: 17-37Crossref PubMed Scopus (443) Google Scholar). A few years ago, an additional motif was identified upstream of motif I that is comprised of several conserved amino acids, including an almost invariant glutamine; this motif has been named the Q motif (Tanner et al., 2003Tanner N.K. Cordin O. Banroques J. Doere M. Linder P. The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis.Mol. Cell. 2003; 11: 127-138Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Although a conserved glutamine residue upstream of motif I is found in most helicases, other characteristic residues of the Q motif are only conserved among DEAD box proteins and, therefore, the Q motif is thought to be specific to DEAD box RNA helicases. Based on structural and biochemical results, the Q motif has been suggested to sense the ATP bound state and, thereby, regulate ATP binding and hydrolysis (Cordin et al., 2004Cordin O. Tanner N.K. Doere M. Linder P. Banroques J. The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity.EMBO J. 2004; 23: 2478-2487Crossref PubMed Scopus (91) Google Scholar). Similar to motif III, the Q motif may not always be essential for function of the protein in vivo, though this may depend on assay conditions (Bernstein et al., 2006Bernstein K.A. Granneman S. Lee A.V. Manickam S. Baserga S.J. Comprehensive mutational analysis of yeast DExD/H box RNA helicases involved in large ribosomal subunit biogenesis.Mol. Cell. Biol. 2006; 26: 1195-1208Crossref PubMed Scopus (41) Google Scholar, Granneman et al., 2006aGranneman S. Bernstein K.A. Bleichert F. Baserga S.J. Comprehensive mutational analysis of yeast DExD/H box RNA helicases required for small ribosomal subunit synthesis.Mol. Cell. Biol. 2006; 26: 1183-1194Crossref PubMed Scopus (31) Google Scholar). For a detailed description of the individual motifs, their structural characteristics, and the intermotif and interdomain interactions of the conserved residues, we refer the reader to the following reviews, which superbly discuss these subjects (Caruthers and McKay, 2002Caruthers J.M. McKay D.B. Helicase structure and mechanism.Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (334) Google Scholar, Cordin et al., 2006Cordin O. Banroques J. Tanner N.K. Linder P. The DEAD-box protein family of RNA helicases.Gene. 2006; 367: 17-37Crossref PubMed Scopus (443) Google Scholar). Despite the fact that the first helicase protein was described over 30 years ago and that since then innumerable RNA and DNA helicases have been identified in all three kingdoms of life, as well as in viruses, we still lack a satisfying understanding of how helicases, especially RNA helicases, function in vivo. In particular, despite the conservation of the helicase core, different helicases clearly function in discrete aspects of RNA metabolism, with questions remaining as to how they are targeted to the right substrates in vivo and how their activities are regulated. Moreover, we have yet to understand the function of the many proteins that are associated with RNA helicases in vivo. Do they function as cofactors to enhance unwinding activity? This scenario is especially intriguing, as many putative RNA helicases lack efficient unwinding activity in vitro compared to the more processive DNA helicases. And how is the energy of NTP binding and hydrolysis translated into strand separation or protein displacement? In this review, we highlight some of the very recent accomplishments in answering these questions, providing new insights into regulation of activity, substrate recognition, as well as our current understanding of the biophysics of the mechanism of RNA helicase action. RNA helicases play essential roles in all facets of RNA metabolism (Table 1). Intriguingly, the majority are involved in ribosome biogenesis (20 out of 39 in yeast) or pre-mRNA splicing (9 out of 39), both biological processes that require a multitude of proteins and small RNAs that together form large dynamic ribonucleoproteins (RNPs). General functions have been assigned to most RNA helicases through combinations of genetic and biochemical experiments; however, in only a few cases have these resulted in a reasonably good understanding of the specific role of these enzymes in vivo, including their possible substrates. The latter is especially facilitated by the availability of in vitro systems to study the enzymatic functions in the context of their biological partners, as is the case for pre-mRNA splicing, but not for eukaryotic ribosome biogenesis. Pre-mRNA splicing is a nuclear event that is essential for producing translatable mRNAs by removing introns from the mRNA precursor. The two transesterification reactions that ligate adjacent exons and remove introns require over 100 proteins and five short nuclear RNAs (snRNAs) that form a dynamic RNP, the spliceosome (reviewed in Brow, 2002Brow D.A. Allosteric cascade of spliceosome activation.Annu. Rev. 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Genetic and biochemical experiments suggest that Sub2 (UAP56 in humans) and Prp5 facilitate binding of the U2 snRNP to the branchpoint, possibly by dissociating proteins binding to the branchpoint or to the U2 snRNA and by rearranging the U2 snRNA structure, the U1 snRNP-mRNA structure, or the mRNA structure at the branchpoint. Consistent with that, the ATPase activity of Prp5 is stimulated by U2 snRNA in vitro, and Prp5 has been suggested to promote the Cus2-regulated conversion of U2-stem IIc into U2-stem IIa during prespliceosome formation (Hilliker et al., 2007Hilliker A.K. Mefford M.A. Staley J.P. U2 toggles iteratively between the stem IIa and stem IIc conformations to promote pre-mRNA splicing.Genes Dev. 2007; 21: 821-834Crossref PubMed Scopus (47) Google Scholar, O'Day et al., 1996O'Day C.L. Dalbadie-McFarland G. Abelson J. The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA.J. Biol. 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