H Box RNA Helicases

Molecular Cell, Vol. 8, 251–262, August, 2001, Copyright 2001 by Cell Press

DExD/H Box RNA Helicases: From Generic Motors to Specific Dissociation Functions N. Kyle Tanner and Patrick Linder1 Department de Biochimie me´dicale Centre Me´dical Universitaire 1, rue Michel Servet CH-1211 Gene`ve 4 Switzerland

Summary RNA helicases of the DEAD box and related DExD/H proteins form a very large superfamily of proteins conserved from bacteria and viruses to humans. They have seven to eight conserved motifs, the characteristics of which are used to subgroup members into individual families. They are associated with all processes involving RNA molecules, including transcription, editing, splicing, ribosome biogenesis, RNA export, translation, RNA turnover, and organelle gene expression. Analysis of the three-dimensional structures obtained through the crystallization of viral and cellular RNA helicases reveals a strong structural homology to DNA helicases. In this review, we discuss our current understanding of RNA helicases and their biological function.

Introduction RNA is a biologically important molecule that is required by all living organisms. It is involved in virtually all aspects of the expression of genetic information, and it plays important regulatory roles to ensure the fidelity of biological reactions. While for many years it has been known that tRNA and ribosomal RNA form specific interactions and structures, it is becoming increasingly clear that this is a general feature of RNAs and that the ability to form these structures is important for the biological activity. Indeed, some RNAs have even been shown to have enzymatic activity by themselves, which is the strongest evidence for the formation of discrete threedimensional structures (e.g., Tanner, 1999). Moreover, because biological processes are dynamic, RNA-RNA, RNA-DNA, and RNA-protein interactions must transiently form and dissociate in a specific order and in an efficient manner. However, just as proteins often require chaperones (Richardson et al., 1998) to assist in folding or refolding the peptide chain into the correct conformation, RNA molecules can require helper molecules to assist in folding or refolding the polynucleotide chain. The ability to unwind folded RNAs or to modify RNARNA interactions has been attributed to RNA helicases. RNA helicases are enzymes that are thought to unwind double-stranded RNA (dsRNA) molecules in an energydependent fashion through the hydrolysis of NTP. They are present in almost all organisms, ranging from bacteria and viruses to humans. Historically, such enzymes 1

Correspondence: [email protected]

Review

are members of the so-called DEAD box family, which was named according to one of the conserved motifs (Linder et al., 1989). After the characterization of the DEAD box family, other related families were defined based on divergent sequence motifs (e.g., Wassarman and Steitz, 1991). At that time, the eukaryotic translation initiation factor 4A (eIF4A) was shown to modify RNA structures in an ATP-dependent fashion, and the genetic characterization of other proteins of the DEAD box family was in agreement with such an RNA helicase function. Soon thereafter, the biochemical demonstration that two DEAD box proteins, p68 and eIF4A, could separate short RNA duplexes confirmed this hypothesis (Hirling et al., 1989; Rozen et al., 1990). Consequently, it was assumed that other DEAD box proteins were also RNA helicases. This was further strengthened by sequence analyses of the RNA helicases that revealed conserved motifs that were shared with the well-characterized DNA helicases (Gorbalenya and Koonin, 1993). In this review, we will summarize our current knowledge about the DExH/D (where x can be any amino acid) proteins and point to recent experimental data and hypotheses. Although sequence comparisons allow us to establish families of related proteins, it is often difficult to assign a specific biological function to many of these proteins, and we still lack a good understanding of their molecular properties. This often reflects the complexities of the biological systems and the difficulties in studying many of these proteins in vitro. Thus, our picture of RNA helicases is undergoing continual revision and modification. Some specific questions that remain unsolved are: How do RNA helicases transmit the energy obtained from ATP hydrolysis into strand separation? Do all these proteins unwind double-stranded nucleic acids, or do they have other activities? What determines substrate specificity? What Is an RNA Helicase? On the sequence level, RNA helicases are identified by the presence of seven to eight conserved motifs (Figure 1) (de la Cruz et al., 1999). These motifs are involved in binding an NTP, generally ATP, and using the energy of hydrolysis to unwind dsRNA. However, many of the putative targets of RNA helicases contain only short basepaired regions, e.g., the proposed targets for the helicases involved in nuclear pre-mRNA splicing (reviewed by Staley and Guthrie, 1998). In these cases, the proteins may be acting as “RNA chaperones” or “maturases” to ensure that the correct interactions are formed or as energy-dependent “facilitators” to drive multistep reactions to completion. Moreover, a continuous dsRNA is rare in biological systems; consequently, a processive dsRNA unwinding activity is generally not needed. This is in contrast to some DNA helicases, which unwind long stretches of dsDNA during replication or repair (Lohman and Bjornson, 1996; Patel and Picha, 2000). A possible exception is the viral helicases, where an RNA helicase may need to resolve dsRNA intermediates formed during transcription or replication

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Figure 1. Conserved Sequence Motifs of RNA Helicases (A) Positions of conserved motifs within eIF4A. The amino- and carboxyl-terminal extensions, before motif I and beyond motif VI, are 45 and 40 amino acids, respectively. This is close to the minimal size seen for an RNA or DNA helicase. Other RNA helicases can have amino- or carboxyl-terminal extensions greater than 500 amino acids. The Rep-like and UvrD-like DNA helicases are characterized by internal insertions, indicated as domain 1b and 2b, as well as a DNA helicase-specific motif IV* (shown in gray). Motif IV is known as IVa in DNA helicases (Korolev et al., 1998). Some RNA helicases derived their name from the characteristics of motif II (e.g., DEAD, DEAH), while others are named after a representative member (e.g., Ski2p, Upf1), as with DNA helicases. This figure is not drawn to scale. (B) Alignments of the conserved motifs. Except for motifs I and II, the conserved motifs historically have been largely defined by the order of appearance of conserved amino acids relative to the Walker NTP binding motifs. Thus, the precise character of the motifs varies between families of helicases and between authors. Amino acids that occupy equivalent positions between families have only been determined where crystal structures are available, as for NS3 and the DEAD box proteins (Caruthers et al., 2000; Lin and Kim, 1999), but ambiguities remain, as for motifs III and IV for Upf1. Amino acids conserved at least 80% of the time are shown as capital letters while those conserved 50%–79% of the time are in lower case. Note that there are additional, highly conserved amino acids outside of the sequences shown that are often family specific.

of the viral RNA (reviewed by Kadare and Haenni, 1997). Nevertheless, such activities are rare and should not be considered a general feature of the RNA helicases. Moreover, some DExD/H helicases may be involved in disrupting or rearranging RNA-protein interactions (Jankowsky et al., 2001; Linder et al., 2001; Schwer, 2001). Thus, the term RNA helicase is controversial, and the reader should consider these proteins as “putative RNA helicases” or “unwindases” as long as their biochemical activity has not clearly been defined. Structural-Functional Properties of Helicases Recent studies, particularly the determination of the crystal structures of various DNA and RNA helicases, emphasize the close relationship of both the conserved motifs and the three-dimensional structures of the enzymatic cores (reviewed by Hall and Matson, 1999; Korolev et al., 1997, 1998; Marians, 2000; Soultanas and Wigley, 2000; Wigley, 2000). This implies that the catalytic cores of the proteins are functionally and mechanistically

equivalent even if they have different biological activities and substrates. While it is outside the realm of this review to describe the well-characterized DNA helicases in any detail, it is useful to briefly outline some of the similarities and differences with RNA helicases to better understand their molecular properties. Some excellent reviews on DNA helicases can be found elsewhere (Lohman and Bjornson, 1996; Patel and Picha, 2000). Gorbalenya and Koonin (1993) were the first to systematically define the relationship between these proteins; they were able to classify the RNA and DNA helicases into different superfamilies (SF) based on the sequence similarities of a few hundred proteins that were available at the time. Moreover, they accurately predicted many of the features seen in the crystal structures. Their classification was based on the presence of conserved motifs that they noted generally occurred in regions that were predicted to be within loops or structural transitions. Thus, for example, UvrD-like and E. coli Rep DNA helicases are classified within SF1, and

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Table 1. Properties of the Conserved Motifs Motif Known or Suggested Function I Ia Ib II

III IV V VI

P-loop; Walker A NTP-binding motif; binds phosphates of NTP1,2 Binds substrate through sugar-phosphate backbone2 Substrate binding2; not as highly conserved and may not always be present Walker B NTP-binding motif; binds ␤ and ␥ phopshate through Mg2⫹; coordinates hydrolysis of NTP with water molecule1,2,3 Binds ␥ phosphate; links NTP hydrolysis with unwinding activity2,4 Substrate binding2; known as IVa in SF1 DNA helicases Binds substrate through sugar-phosphate backbone; may interact with NTP2 Binds ␥ phosphate; converts NTP binding/hydrolysis with domain 1 and 2 movement2,5

1

(Smith and Rayment, 1996; Walker et al., 1982) Based on analyses of crystal structures (Caruthers et al., 2000; Koroleve et al., 1998; Lin and Kim, 1999) 3 (Fry et al., 1986) 4 Mutations of motif can dissociate NTP hydrolysis from unwinding activity (Pause et al., 1993; Schwer and Meszaros, 2000). 5 Mutations of motif can affect substrate binding and NTPase activity (Linder et al., 2000; Hall and Matson, 1999, and reference therein). 2

the DEAD-like and DEAH-like RNA helicases are placed in SF2. The E. coli DnaB and Rho helicases are classified into SF4 and SF5, respectively. Most DExD/H RNA helicases are classified within SF2, but many viral RNA helicases are in SF1. All of these proteins are characterized by the Walker A and B motifs (motifs I and II in SF1 and SF2), which is an NTP binding motif that is found in a wide variety of NTPase enzymes (Smith and Rayment, 1996; Walker et al., 1982). These two motifs are necessary but not sufficient to define a helicase. Most DExD/H proteins seem to use ATP, but many are promiscuous in the nucleotide used in vitro (e.g., Wardell et al., 1999). In addition, SF1 and SF2 proteins have five to six other motifs in common (Figure 1 and Table 1), and both are generally considered to be active as either monomers or dimers. The other superfamilies (SF3, SF4, SF5) are probably active as hexameric complexes, and they are clearly seen as “doughnuts” by electron microscopy (reviewed by Patel and Picha, 2000); these latter helicases are more distantly related to the RNA helicases, and consequently our further discussion will be limited to SF1 and SF2. Finally, it should be noted that the classification of proteins within SF1 and SF2 was based on limited data and was considered preliminary (Gorbalenya and Koonin, 1993). The recent explosion of sequences data in the databases, plus more sophisticated alignment tools, means that more detailed analyses can be made. Such alignments, plus the solved crystal structures, indicate that these proteins are more closely related than their superfamily designations would suggest. Mutations have been made in a number of the different helicases (reviewed by Hall and Matson, 1999; Linder et al., 2000). In general, these mutations are consistent with the motifs being involved in binding the NTP and translating NTP hydrolysis into conformational changes that are associated with unwinding or translocation activity. However, because the character and severity of

mutations within any particular motif can vary widely depending on the properties of the protein under study and its biological function, it is sometimes difficult to make direct comparisons of mutations between subfamilies of helicases. Fortunately, the recently solved crystal structures of the hepatitis C virus (HCV) NS3 protein (Cho et al., 1998; Kim et al., 1998; Yao et al., 1997), the DEAD box protein from the archaebacterium Methanococcus jannaschii (Story et al., 2001), and fragments of eIF4A from yeast (Benz et al., 1999; Caruthers et al., 2000; Johnson and McKay, 1999) have permitted a direct structural comparison with the solved crystal structures of the SF1 DNA helicases (reviewed by Korolev et al., 1998; Bird et al., 1998; Korolev et al., 1997; Lin and Kim, 1999). It is clear that the core structures of these SF1 and SF2 proteins can be largely superimposed, and therefore it is possible to extrapolate studies of one protein to the others. The strong structural conservation implies an equally strong functional conservation. However, this can be misleading as not all families have been shown to have dsDNA or dsRNA unwinding activity in vitro; e.g., members of the Snf/Swi family of proteins are involved in chromatin rearrangements and may be better defined as translocases (Havas et al., 2000 and references therein). Moreover, most RNA helicases are incapable of unwinding long double-stranded regions in vitro, as opposed to the processive activity of some of the DNA helicases. Nevertheless, these differences probably reflect the properties of additional, family-specific sequences (either internal or terminal extensions) that form separate structural domains, and consequently it is useful to seek a minimal structure that is consistent with the activity seen in all of these proteins. A search of protein databases reveals that eIF4Alike DEAD box proteins are consistently the smallest proteins that contain all of the helicase motifs (N.T., A. Gattiker, and P.L., unpublished data). They can have as few as ⵑ45 amino acids upstream from motif I and ⵑ40 amino acids downstream from motif VI. This is useful to note because eIF4A is also the smallest protein shown to have unwinding activity in vitro (Rogers et al., 2001; Rozen et al., 1990); thus, it represents a minimal, helicase, enzymatic core. It is similar in size to the recently crystallized but biologically uncharacterized DEAD box protein from M. jannaschii (MjDEAD) (Story et al., 2001). This conclusion is further supported by the in vitro helicase activity of an amino-terminal-deletion fragment of the DEAH box protein Prp22p that contains only 40 amino acids upstream from motif I. Moreover, it has in vivo activity when the deleted amino terminus, which provides specificity, is coexpressed in trans (Schneider and Schwer, 2001). On this basis, full-length MjDEAD (Story et al., 2001) and the core structures of PcrA (Velankar et al., 1999) and NS3 (Kim et al., 1998) are compared in Figure 2A. The complete structure of eIF4A was constructed from the individually determined structures of domains 1 and 2 and then assembled based on the structure of NS3 (Caruthers et al., 2000). It is similar to NS3 except for a short amino-terminal extension or cap, which is very important for DEAD box protein activity in vivo (J. Banroques, O. Cordin, N.T., and P.L., unpublished data). This is seen in the three-dimensional structure of MjDEAD

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Figure 2. Three-Dimensional Structures of RNA and DNA Helicases (A) The core structures, as defined by eIF4A, are shown as ribbons and cords; positions of insertions and extensions are indicated with arrows. An ATP is shown bound within the P loop of PcrA; a sulfate is shown in the equivalent position of NS3 and MjDEAD. The motifs are shown in different colors (as in Figure 1) and indicated by roman numerals. Amino acids in motif IV of NS3 were not all resolved in the crystal structure and are shown as a gap. To facilitate comparisons, motifs are aligned relative to conserved amino acids defined by Lin and Kim (1999) and

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protein, where the amino-terminal cap forms a helixloop-helix-loop. Another difference is that motif III in NS3 directly links the two domains. In the other crystal structures (DNA and RNA), motif III occurs within the first loop of a loop-helix-loop linker, where the helix interacts with domain 1 and the second loop spans the cleft between domains. A detailed summary and comparison of the motifs and structural features of SF1 and SF2 has been made (Hall and Matson, 1999; Korolev et al., 1997, 1998; Lin and Kim, 1999; Marians, 2000; Soultanas and Wigley, 2000, 2001; Wigley, 2000). Because of space limitations, we will cite their reviews to summarize the various features described here; references to the original articles can be found therein. While the cores of the DNA and RNA helicases as shown in Figure 2A can be largely superimposed, they do differ in several important respects. RNA helicases often have very large amino- or carboxyl-terminal extensions that can be longer than 500 amino acids. Figure 2B shows the complete proteolytic fragment of NS3 that contains helicase activity in vitro, where the carboxyl terminus forms domain 3. The amino-terminal third of the protein, which contains a serine protease activity, was removed to facilitate crystallization. Certain families of SF1 and SF2 DNA helicases (e.g., RecQ and Pif1) also can have long terminal extensions, but the Rep-like and UvrD-like DNA helicases tend to be short. They are characterized by large insertions within the core sequences of domain 1 and 2, thus forming domains 1b and 2b. These domains occupy a position within the structures that is more or less equivalent to domain 3 of NS3. It is likely that these additional domains confer other functionality to the protein, perhaps by making it more processive or by facilitating the denaturation of doublestranded regions, as proposed for PcrA (Soultanas et al., 2000). Proteins lacking these domains may acquire the activity through their association with other factors. For example, the unwinding activity of eIF4A is greatly improved by the addition of eIF4B (Rozen et al., 1990); it could function as an intermolecular domain 3. Further evidence for this is that, within certain families of RNA helicases, domain 3 appears to be structurally conserved, and that, at least in some cases, it is more sensitive in vivo to terminally derived deletions than the amino-terminal domain (Schneider and Schwer, 2001, and references therein). However, the length of domain 3 varies widely between RNA helicases, and its exact role is still speculative. Moreover, we have found for several DEAD box proteins from yeast that the carboxylterminal domain can be removed without destroying in vivo activity (N.T., J. Banroques, M. Doere, and P.L., unpublished data). The amino-terminal extensions of RNA helicases can have other activities associated with them, e.g., serine protease for NS3 (Gallinari et al., 1998),

nuclear signaling, or the conferring of specificity through interactions with other proteins and factors (Schneider and Schwer, 2001; Wang and Guthrie, 1998). The most notable feature of the enzymatic core is that it consists of two discrete domains connected by a linker region that forms a cleft into which an NTP can bind. The conserved motifs are largely facing into this cleft and are involved in substrate binding, NTP binding, and coordinating NTP hydrolysis with helicase and translocation activities (see Table 1). Motifs I and II are NTP binding motifs first identified by Walker et al. (1982; Walker motif A and B). Motif I forms a loop structure, also called the P loop, that forms a pocket to bind the phosphates of NTP. Motif II forms interactions with the ␤ and ␥ phosphates through a coordinated Mg2⫹ (Fry et al., 1986). It is probably involved in the hydrolysis of the NTP by the addition of a water molecule. Motifs Ia, Ib, IV, and V are probably involved in substrate binding, while motifs III and VI somehow link NTP binding and hydrolysis with conformational changes needed for helicase activity. Indeed, mutations within motif III can dissociate NTP hydrolysis from unwinding activity (Pause and Sonenberg, 1992; Schwer and Meszaros, 2000). Interestingly, motif VI was originally defined as the substrate binding site; this was based on mutations within the motif that affected substrate binding of eIF4A (Pause et al., 1993). Indeed, the authors of the first crystal structures of NS3 were able to model an oligomer within the cleft between domains 1 and 2 (Cho et al., 1998; Yao et al., 1997). This appears not to be the case. For example, mutations in motif VI of NPH-II affect ATP hydrolysis but not RNA binding (Gross and Shuman, 1996). In NS3, the RNA (or DNA) substrate appears to be bound in a second cleft formed between domains 1–2 and domain 3. In DNA helicases, this cleft is formed by domains 1a–2a and domains 1b–2b. UvrD-like proteins contain a second motif, shown as motif IV* in Figure 1A, that forms interactions with the ␥ phosphate of the bound NTP; its role is duplicated by additional interactions of motif VI in RNA helicases (Lin and Kim, 1999 and references therein). Finally, there are a number of interactions with isolated conserved amino acids. Some of these are “gatekeepers” that intercalate or stack on the bases of the bound substrate and thereby function like the teeth of a ratchet to hold the substrate in position. This provides an appealing model for helicase activity, because the distance between the amino acid “teeth” can vary with NTP-dependent closing and opening of the cleft between domains 1 and 2 (see below under “Models for Helicase Activity”). Substrate Specificity and Helicase Activities In general, RNA helicases have shown very little substrate specificity when analyzed in vitro. Moreover, none

Caruthers et al. (2000). Note that Rep-like and UvrD-like helicases have an extra motif IV, indicated as IV*, that is not found in the RNA helicases. Only a portion of the DNA substrate is shown for PcrA. (B) The fragment of NS3 that has RNA helicase activity in vitro. The amino-terminal one-third of the protein, which contains a serine-protease activity, was removed to facilitate crystallization. The PDB coordinates used were 1A1V for HCV NS3 (Kim et al., 1998), 3PJR for Bacillus stearothermophilus PcrA (Velankar et al., 1999), and 1HV8 for Methanococcus janaschii MjDEAD (Story et al., 2001), and they were obtained from the authors or through Research Collaboratory for Structural Bioinformatics (RCSB; Berman et al., 2000). The PDB coordinates were modeled on the Macintosh with Swiss-PdbViewer (Guex and Peitsch, 1997); http://www.expasy.ch/spdbv/) and the images rendered with POV-Ray (http://www.povray.org/).

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of the substrate-protein interactions seen in the crystal structure of NS3 involve the recognition of specific nucleotide bases (Kim et al., 1998). Instead, interactions involve primarily base stacking and the sugar-phosphate backbone, and this is true for DNA helicases as well (Caruthers et al., 2000; Lin and Kim, 1999). This provides a mechanism for distinguishing between RNA, with its 2⬘ hydroxyl, from DNA but not for distinguishing between sequences. This is not entirely surprising for proteins involved in unwinding double-stranded nucleic acids; any sequence specificity might hinder the movement of the helicase along the strand. Nevertheless, most of these proteins are highly specific for substrates in vivo; e.g., ribosomal biogenesis or splicing (see Processes Requiring RNA Helicases). This is probably conferred by the context in which the substrate appears and most likely on associated protein factors. DNA helicases are often found complexed with factors involved in DNA repair or replication (Lohman and Bjornson, 1996; Patel and Picha, 2000). RNA helicases are often found within large ribonucleoprotein complexes (translation initiation, ribosomal precursors, spliceosomes, etc). Thus, the “catalytic cores” of these proteins could have a generic substrate binding affinity, while specificity is conferred by the interactions of the flanking sequences and sequences on the external surface of the cores with other proteins. Indeed, the flanking sequences, when overexpressed alone, can have a dominant-negative phenotype in yeast, suggesting that they sequester other factors or that they assemble into nonfunctional complexes (Wang and Guthrie, 1998). Moreover, the amino-terminal domains of Prp16 and Prp22 are needed for localization within the spliceosome (Schneider and Schwer, 2001; Wang and Guthrie, 1998). Thus, to better understand the helicase properties, it is important to study the activity with the associated factors. This is possible where complexes can be reconstituted in vitro, as for translation initiation and nuclear pre-mRNA splicing. Another possibility, as suggested by the presence of the S1 motif in the DEAH box proteins involved in splicing (Company et al., 1991), is that the flanking sequences form independent interactions with the substrate. This is further supported by the in vitro substrate specificity of the E. coli DEAD box protein DbpA (FullerPace et al., 1993; Pugh et al., 1999). In this case, the carboxyl-terminal domain is proposed to form specific interactions with hairpin 92 of the 23S rRNA (Tsu et al., 2001). In many cases, it has been difficult to demonstrate RNA unwinding activity in vitro. For eIF4A, the original activity depended on the addition of eIF4B, which is another translation initiation factor; only recently has eIF4A by itself been shown to unwind dsRNA, albeit only for duplexes with limited stability (Rogers et al., 2001). Interestingly, only mammalian eIF4B stimulates yeast and mammalian eIF4A; the yeast eIF4B protein has no effect (Altmann et al., 1995; Rozen et al., 1990). In nearly all of the different helicase assays, a large excess of protein over substrate is required. This could reflect the absence of cofactors and a low intrinsic affinity for the substrate, or it could reflect partial inactivation of the purified proteins. For example, the NS3 protein is largely insoluble when expressed in E. coli; it must first undergo a denaturation-renaturation step to regain its activity (e.g., Khu

et al., 2001). Evidence for a “helper” protein has been obtained for Dbp5p, which is involved in RNA export (Schmitt et al., 1999Q1; Tseng et al., 1998). A recombinant Dbp5p has RNA-stimulated ATPase activity, but it shows no unwinding activity in vitro. However, Dbp5p obtained by immunoprecipitation from a cell extract does have unwinding activity, suggesting the coprecipitation of an essential cofactor. As with DNA helicases, most RNA helicases unwind dsRNA in either a 5⬘ to 3⬘ or a 3⬘ to 5⬘ direction when tested in vitro (de la Cruz et al., 1999). This is determined by providing double-stranded substrates with either 5⬘ or 3⬘ single-stranded overhangs. These overhangs enable the helicase to load on the substrate; most RNA helicases have a very low initial affinity for dsRNAs. However, some helicases do not have polarity when tested in vitro; e.g., eIF4A will unwind dsRNA with either a 5⬘ or 3⬘ single-stranded overhang (Rozen et al., 1990). The size of the substrate binding site is estimated to be 8–10 bases based on the crystal structures of DNA and RNA helicases (Kim et al., 1998; Korolev et al., 1997; Velankar et al., 1999). This is consistent with the size of the single-stranded overhangs used for artificial substrates. Estimates for the NTP-dependent step size have been made for NS3 and various DNA helicases, which range from four to five bases to as little as one (reviewed by Marians, 2000; Soultanas and Wigley, 2000; Wigley, 2000). The putative substrates for the best characterized RNA helicases contain duplex regions that are generally below 10 bp. This is roughly the size of the binding site, and it would require as little as a single step for unwinding. Thus, a processive activity, as for some DNA helicases, is generally not required. In these cases, the RNA helicase may be acting more like a temporary clamp to prevent RNAs from reassociating, thereby allowing other RNA-RNA or RNA-protein interactions to occur. This would ensure the directionality and efficiency of multistep reactions, such as pre-mRNA splicing or ribosomal biogenesis. It is also consistent with the observation that helicases are only transiently associated with the spliceosome (Neubauer et al., 1998). However, a processive helicase activity has been demonstrated for the viral RNA helicase NPH-II from vaccinia virus (Jankowsky et al., 2000). This DExH helicase can unwind long dsRNA (up to 83 bp) in the presence of large amounts of competitor RNA that acts to “trap” dissociated helicase protein; it unwinds about 6 bp per kinetic step (not necessarily equivalent to a single physical step), which is similar to that determined for UvrD (Ali and Lohman, 1997). However, the activity diminished with increasing duplex size, suggesting that additional factors may be needed to stabilize the protein on the substrate. Models for Helicase Activity A number of models have been proposed to explain helicase activity, but the two most widely discussed are the active rolling model and the modified inchworm model (reviewed by Lin and Kim, 1999; Lohman and Bjornson, 1996; Marians, 2000; Soultanas and Wigley, 2000, 2001). Both were originally proposed for the DNA helicases, but they are equally applicable to the RNA helicases. Both models are consistent with various ex-

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Figure 3. Models of Helicase Activity The active rolling model requires at least a dimerized helicase in which each monomer has a different conformational state and affinity for single-stranded and double-stranded nucleic acids. These conformations vary with NTP binding and hydrolysis. It proposes a step size that is equivalent to the binding-site size or 8–10 bases. Note that the handiness shown is used to distinguish between subunits, and it should not be inferred that the subunits are mirror images of each other. The inchworm model works with a monomer or oligomer state. In this case, the distance between domains 1 and 2 vary with NTP binding and hydrolysis. Specific amino acids, shown as the feet of the inchworm (or caterpillar) form nonspecific interactions with the sugar-phosphate backbone, or they stack or intercalate against the bases; these latter interactions act as teeth to help maintain the positions of domains rather like the sprocket of a ratchet. This provides a mechanism to ensure that the helicase moves with a specific polarity. These models are described in more detail in the text.

perimental observations, but neither one fully explains all of them. The fundamental difference between the two models is that the active rolling model requires at least a dimer to be functional while the inchworm will work with both monomers and oligomers. For the active rolling model, the dimers are in two different conformational states (Figure 3). One state has a high affinity for ssRNA/ DNA, and the other has a higher affinity for dsRNA/DNA. The conformational states vary with the binding and hydrolysis of NTP. In this way, the helicase dimer acts “hand-over-hand” to move along the RNA/DNA and unwind the double-stranded regions. In the inchworm model, the monomer undergoes conformational changes associated with the binding and hydrolysis of NTP that move domain 1 and domain 2 closer or further apart relative to each other (Figure 3). The substrate is bound by both domains. In the crystal structures of NS3 and PcrA (Kim et al., 1998; Velankar et al., 1999), specific amino acids are seen intercalating or stacking on the bases, and they act like gatekeepers, equivalent to the teeth of a ratchet, to hold the substrate in position relative to the two domains. Thus, for the inchworm model to work and to provide polarity to helicase movement, each ratchet tooth must temporarily

and sequentially dislocate from the substrate. This model really does invoke the image of an inchworm moving down the strand. In both models, the unwinding of the dsRNA/DNA would require conformational alteration of the substrate, perhaps by forcing the double-strand phosphate backbone to assume an energetically unfavorable conformation. For the active rolling model, this role would be played by NTP-dependent differences in the substrate binding site. For the inchworm model, this function is most easily envisioned for domain 3 of RNA helicases and domains 1b–2b of DNA helicases. Indeed, in one crystal structure of PcrA, the dsDNA is seen pressed against domains 1b–2b at a sharp angle relative to the bound ssDNA strand (Velankar et al., 1999). The unidirectional closing and opening of domains 1a and 2a with NTP binding and hydrolysis could put additional contorsional stress on the Watson and Crick hydrogen bonds and thereby force the double-stranded region open (Velankar et al., 1999). Further evidence for this comes from mutations within domains 1b and 2b of PcrA that can uncouple DNA translocation from unwinding activity (Soultanas et al., 2000). To date, the inchworm seems to best explain RNA helicase activity. However,

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there is some controversy about whether NS3 functions as a monomer or oligomer, and there is evidence that E. coli Rep is only functional as an oligomer (Cheng et al., 2001). To further complicate things, the Brr2 subfamily of RNA helicases contains two helicase cores, although mutational analysis suggests that only one is functionally required (Kim and Rossi, 1999). While none of this is inconsistent with the inchworm model, it does suggest that the actual mechanism may be more complicated than the model implies. Processes Requiring RNA Helicases Proteins of the DExD/H box and related families are involved in a variety of biological processes. These include transcription, ribosome biogenesis, pre-mRNA editing and splicing, RNA export to the cytoplasm, translation initiation and termination, RNA degradation, organelle gene expression, and virus propagation. Within these processes, RNA helicases may be highly specific and rearrange only limited regions of the RNA, or they may processively unwind long dsRNA. Some may even not be RNA helicases per se but rather RNPases. The involvement of putative RNA helicases in biological reactions has been extensively reviewed in the past, and therefore we will not discuss them in any detail (de la Cruz et al., 1999; Eisen et al., 1995; Fuller-Pace, 1994; Jankowsky and Jankowsky, 2000; Linder et al., 2000; Muchardt and Yaniv, 1999; Staley and Guthrie, 1998; Venema and Tollervey, 1999). Nevertheless, it is useful to give a brief overview of some of these processes to better appreciate the myriad of activities. In the following discourse, we will describe the various helicase-dependent modifications that occur from RNA transcription to RNA degradation. Ribosomal biogenesis and pre-mRNA splicing will be given particular emphasis because they are better characterized genetically and biochemically. Transcription initiation relies in part on the conformation of chromatin. Several proteins of the Snf/Swi family, which is related to the RNA helicases, have been shown to be involved in transcriptional regulation through the modulation of chromatin structure or through interactions with the assembly of the transcription-initiation complex (see Muchardt and Yaniv, 1999). In addition to the Snf/Swi proteins, a subfamily of the DEAH family is involved in transcriptional regulation. Within this subfamily, the mle protein from Drosophila melanogaster has been shown to be involved in dosage compensation (Lee et al., 1997). Eukaryotic ribosome biogenesis is a highly dynamic process involving 78 ribosomal proteins, four rRNA molecules, and a variety of trans-acting factors, including a large number of small nucleolar guide RNAs (snoRNAs; Figure 4A; reviewed by Kressler et al., 1999; Venema and Tollervey, 1999). Thus, it is not surprising that 17 RNA helicases from the yeast Saccharomyces cerevisiae have been implicated in ribosome biogenesis and that most, if not all, have highly conserved counterparts in other organisms. Although the exact roles of these helicases are not known, the phenotypes obtained upon depletion or inactivation of the different helicases in yeast give an indication of their function. By this criterium, eight helicases have been shown to be involved in early processing steps because their inactivation gives a deficit of 40S subunits. Nine helicases are involved in

steps affecting 60S subunit biogenesis. We and others have shown that most of these helicases are essential and highly specific; i.e., they cannot substitute for each other, even when overexpressed. What, then, could be the function of all these helicases in ribosome biogenesis? A large part of the final mass of a eukaryotic ribosome is RNA. During biogenesis, this rRNA associates with many trans-acting factors, including the snoRNAs involved in rRNA modification. Thus, RNA helicases may be required to facilitate individual steps in the processing pathway or to act as energy-dependent enzymes to drive the reaction to completion. With over 100 snoRNAs in yeast and even more in mammalian cells implicated in ribosome biogenesis, there are many potential helicase substrates, although only a few have been found genetically or physically associated with snoRNAs in yeast (Colley et al., 2000; Liang et al., 1997; Venema et al., 1997). Genetic and biochemical analysis of pre-mRNA splicing by the spliceosome has greatly contributed to the interest of RNA helicases, and their role in splicing has been extensively discussed (Staley and Guthrie, 1998). This has been facilitated by the development of in vitro reconstitution assays and by nondenaturing polyacrylamide gels that resolve assembly intermediates. Pre-mRNA splicing involves two transesterification reactions, where the net change of energy (⌬H) of the phosphodiester bonds is zero. Nevertheless, there is an absolute energy requirement, generally ATP, and this has been attributed largely to the activity of RNA helicases. The spliceosome is a large ribonucleoprotein (RNP) particle consisting of five small nuclear RNAs (snRNA) and more than 50 proteins. It is also a very dynamic machine involving the sequential alteration of RNA-RNA and RNA-protein interactions. For example, the U1 and then U6 snRNAs bind the pre-mRNA at the 5⬘ splice site in a sequential order. Other interactions are also supplanted by various snRNAs during the course of the assembly and reaction pathway (Figure 4B). From genetic and in vitro assays, the sites of action of individual helicases in splicing have been established fairly well. Eight DExD/H RNA helicases are implicated, and most of them are essential in yeast (Staley and Guthrie, 1998). As in ribosome biogenesis, RNA helicases probably rearrange short regions of basepairing between the snRNAs and the pre-mRNA. However, recent genetic experiments also suggest that RNA helicases alter RNA-protein interactions as well and therefore that they have RNPase activity. Indeed, two groups showed that mutations in nonhelicase proteins or snRNAs of the snRNP complexes could render some of the RNA helicases dispensable (Chen et al., 2001; Kistler and Guthrie, 2001). In both cases, weakening the interactions of the RNP complexes would be sufficient to bypass the requirement for a specific RNA helicase without drastically affecting the growth of the cell. The notion of an RNA-protein dissociating machinery is not far-fetched, and it has been proposed on theoretical grounds (Lorsch and Herschlag, 1998; Staley and Guthrie, 1998). Eukaryotic translation initiation of most cellular mRNAs begins with the association of the 40S ribosomal subunit and then scanning for the initiation codon. This process has been known for a long time to be inhibited by secondary structures within the 5⬘ untranslated region (5⬘UTR) of mRNAs (Pause and Sonenberg, 1993).

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Figure 4. Ribosome and Spliceosome Assembly Are Highly Dynamic Processes (A) Eukaryotic ribosome is highly conserved from yeast to humans. In yeast, the rDNA is transcribed as a 35S pre-rRNA precursor. This precursor is modified by methylation and pseudo-uridylation reactions. The modifications and the early cleavage reactions at sites A0, A1, and A2 take place in a large 90S preribosomal particle. Cleavage at A2 allows splitting of the 90S particle into two precursors that will mature into the 40S and 60S subunits. Maturation of the 40S subunit involves export into the cytoplasm and maturation of the 20S pre-rRNA into the mature 18S rRNA. The maturation of the 60S subunit involves several processing reactions resulting in the mature 5.8S and 25S rRNAs. Endonucleolytic and exonucleolytic reactions are indicated by red and green arrows, respectively. RNA helicases (yellow boxes) of the DExD/H families play essential roles in these maturation steps. The helicases Has1p, Mak5p, and Drs1p have not been attributed to a particular step. (B) Nuclear pre-RNA splicing involves several steps that depend on the activity of DExD/H RNA helicases (yellow boxes). For splicing to occur, several snRNP complexes (green circles) transiently and sequentially associate with the pre-mRNA.

Based on these observations, it was suggested that eIF4A, the prototype of DEAD box proteins, is required for unwinding such secondary structures. Recent work by the Sonenberg laboratory provides evidence supporting this hypothesis by using dominant-negative mutants of eIF4A in a cell-free translation system (Svitkin et al., 2001). Under these conditions, translation initiation is sensitive to the extent of “secondary structures” within the mRNA. Nevertheless, strong evidence for unwinding activity of eIF4A in vivo is still lacking. Another DEAD box helicase, Ded1p, has been isolated through a screen of genetic interactions with eIF4E in yeast. Deletion analysis and temperature-sensitive mutations within the DED1 gene have clearly shown that Ded1p is a general translation-initiation factor (Chuang et al., 1997; de la Cruz et al., 1997). Moreover, experiments from Schizosaccharromyces pombe indicate that normal expression levels of Ded1p are required for the translation of several proteins critical for the cell cycle (Grallert et al., 2000). RNA helicases also are required for translation termination and for the programmed destruction of nonsense-codon-containing mRNAs. Upf1p is an RNA helicase belonging to the SF1 family of helicases implicated in DNA and RNA metabolism and is conserved from yeast to humans. Deletion of the UPF1 gene results in

increased readthrough at stop codons and stabilization of nonsense-codon-containing mRNAs (nonsense-mediated decay [NMD]). Upf1p has been shown to be an RNA helicase with 5⬘ to 3⬘ unwinding activity in vitro. The two activities assist in translation termination and degradation of mRNA, and they can be separated by mutational analysis in yeast (Weng et al., 1996). It has been shown that deletion of an N-terminal cysteine/ histidine-rich region affects only translation termination, whereas mutations in motifs II or VI affect mRNA turnover but not translation termination. Interestingly, a mutation in motif I that abolishes ATP binding is deficient in translation termination and NMD. Although the enzymatic activity for translation termination is dependent on the conserved helicase motifs (as measured by readthrough at nonsense codons), this activity and mRNA turnover are two functions that can clearly be separated; i.e., the readthrough per se does not stabilize the nonsense-codon-containing transcript. Conclusions and Perspectives From the above descriptions, it is clear that DExD/H RNA helicases are a widely dispersed group of proteins found in virtually all biological processes requiring

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RNAs. They are associated with alterations of all the different interactions found in RNA, including RNA-RNA, RNA-DNA, and RNA-protein. RNA helicases are energydependent translocation motors that catalyze conformational changes and drive multistep reactions requiring sequential rearrangements of RNA interactions. Thus, RNA helicases can also be considered to be RNA chaperones, maturases, unwindases, and even RNPases. However, all of these activities depend on the ability of the helicase to transiently bind an RNA substrate in an NTP-dependent fashion, and thus they all can be considered as molecular “bookmarks” or temporary “clamps” that permit the rearrangement of RNA interactions in a controlled order. Although many putative RNA helicases have been biochemically and genetically analyzed, a clear molecular role of these enzymes, particularly in vivo, is still missing. Thus, the in vitro reconstitution systems, like translation initiation and pre-mRNA splicing, will do much to reveal the properties of these proteins. Until recently, the medical interest in RNA helicases has lagged behind the DNA helicases, which often are associated with various genetic maladies or are diagnostic of diseased states. However, RNA helicases are essential for bacterial and viral propagation, and consequently they are good targets for antibacterial and antiviral drugs. Moreover, because RNA helicases are involved in a wide variety of cellular activities, they could also be the targets for anticancer drugs or prophylactic agents. Clearly, we still have much to learn about these proteins, and the coming years will be exciting in the helicase field. Acknowledgments We are most grateful to Josette Banroques, Frances Fuller-Pace, Eckhard Jankowsky, Dieter Kressler, Timothy Lohman, Randall Story, Beate Schwer, and the referee for discussions and comments on the manuscript. We would also like to thank Dave McKay, Ulrich Baumann, and Randall Story for making the PDB coordinates available. Work in the authors’ laboratory is supported by a grant from the Swiss National Science Foundation (to P.L.). References Ali, J.A., and Lohman, T.M. (1997). Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275, 377–380. Altmann, M., Wittmer, B., Methot, N., Sonenberg, N., and Trachsel, H. (1995). The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity. EMBO J. 14, 3820–3827. Benz, J., Trachsel, H., and Baumann, U. (1999). Crystal structure of ATPase domain of translation initiation factor eIF4A from Saccharomyces cerevisiae—the prototype of the DEAD box protein family. Structure Fold Des. 7, 671–679. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N., and Bourne, P.E. (2000). The Protein Data Bank. Nucleic Acids Res. 28, 235–242. Bird, L.E., Subramanya, H.S., and Wigley, D.B. (1998). Helicases: a unifying structural theme? Curr. Opin. Struct. Biol. 8, 14–18. Caruthers, J.M., Johnson, E.R., and McKay, D.B. (2000). Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. Proc. Natl. Acad. Sci. USA 97, 3080–3085. Chen, J.Y.-F., Stands, L., Staley, J.P., Jackups, R.R., Jr., Latus, L.J., and Chang, T.-H. (2001). Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Mol. Cell 7, 227–232.

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