dead-box proteins: the driving forces behind rna ... - Semantic Scholar

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DEAD-BOX PROTEINS: THE DRIVING FORCES BEHIND RNA METABOLISM Sanda Rocak and Patrick Linder RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism. They are associated with many processes ranging from RNA synthesis to RNA degradation. DEAD-box proteins use the energy from ATP hydrolysis to rearrange inter- or intra-molecular RNA structures or dissociate RNA–protein complexes. Such dynamic rearrangements are fundamental for many, if not all, steps in the life of an RNA molecule. Recent biochemical, genetic and structural data shed light on how these proteins power the metabolism of RNA within a cell.

HELICASE

An enzyme that unwinds double-stranded nucleic acids in an energy-dependent manner.

Departement de Biochimie Médicale, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Genève 4, Switzerland. Correspondence to P.L. e-mail: Patrick.Linder@ medecine.unige.ch doi:10.1038/nrm1335

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In textbooks, a nascent RNA molecule is often presented as a single-stranded molecule that emerges from the RNA polymerase complex. Although illustrative, this is probably rather different from the fate of an RNA molecule within a cell, where it will encounter a variety of different proteins and other nucleic acids that interact with the nascent or mature RNA. In addition, RNA molecules have a high propensity to form intramolecular interactions. Some of these interactions are necessary for maturation of the RNA or for its activity. However, other intra- or inter-molecular interactions might be deleterious for its function. So, many, if not all, RNA molecules require proteins and other nucleic acids to assist them in the maturation process. Different proteins interact with RNA molecules and help them fold properly and mature. These dynamic processes are probably best illustrated in pre-mRNA splicing or ribosome biogenesis, where continuous rearrangements of the ribonucleoprotein (RNP) complexes are necessary to obtain a mature product. One class of RNA-binding proteins that prevents singlestranded RNA from undergoing non-productive rearrangements, or from inappropriately binding other proteins, is represented by the RNA helicases. These proteins might also help to rearrange RNA into, or within, larger complexes. On the basis of conserved sequence motifs, RNA helicases can be grouped into different families that are classified, together with DNA helicases, as belonging to one of various superfamilies. The

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DEAD-box protein family of RNA helicases belongs to superfamily II (REF. 1) and is by far the largest family of RNA helicases. Here we will summarize the roles that DEAD-box proteins have in various processes. Although they are required for very different processes involving RNA, the enzymes from the DEAD-box family all contain a structurally conserved core element. This core element contains all the conserved motifs that are required for HELICASE activity. Most of the motifs have been characterized by genetic and biochemical analyses and their roles have been confirmed and extended by crystallographic data. The specificity of these proteins for different processes relies mainly on the flanking, less conserved, sequences. In light of several studies on the unwinding of different DEAD-box proteins and on the basis of our knowledge of the processes they are involved in, we will present models for how ATP-binding and hydrolysis by the core element could be responsible for the enzymatic activity of these proteins — that is, the displacement of duplex RNA or RNA–protein complexes. What makes a DEAD-box RNA helicase?

The DEAD-box protein family was first described 15 years ago2. At that time, the alignment of eight proteins that are required for a variety of processes in different species (yeast, mouse, bacteria and fruitfly) was sufficient to define eight conserved motifs, as well as a few conserved, but isolated, residues. One of the motifs

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Box 1 | The conserved motifs of DEAD-box proteins and their interaction with ATP Substrate

Domain 1 N

F

Domain 2

Q

I

Ia

Ib

II

III

IV

V

VI

GAxxPoxxQ

AxxGxGKT

PTRELA

TPGR

DEAD

SAT

LIV

ARGID

HRxGRxGR

C

NH2 N

N N

Mg2+

N O P

O OH

P

P

OH

A sequence alignment of the DEAD-box proteins has revealed nine conserved motifs. On the basis of mutational analyses and structural data, roles for many of the consensus elements have been postulated. The Q-motif and motifs I and II (Walker motif A and B, respectively93) bind ATP and are required for its hydrolysis. The highly conserved glutamine in the Q-motif forms hydrogen bonds with nitrogen atoms at positions 6 and 7 of the adenine, whereas the adjacent aromatic residue stacks with the adenine base. Both glutamine and serine/threonine (depicted as ‘o’ in the diagram) from the Q-motif form hydrogen bonds with conserved residues of motif I (REF. 3). It was proposed that the Q-motif, as well as the upstream phenylalanine residue, regulate ATP binding and hydrolysis4. Motif I (AxxGxGKT) forms a loop structure (P loop) that accommodates the α- and β-phosphates of ATP; the lysine residue interacts with phosphates of MgATP or MgADP, whereas the threonine residue interacts with Mg2+. Motif II (or the DEAD motif) forms interactions with the β- and γ-phosphates through Mg2+ (REFS 12,18,94) and is required for ATP hydrolysis. In the eIF4A structure15, motif II forms hydrogen bonds with the conserved residues of motif III in domain 1. Motif III links the ATP binding and hydrolysis to conformational changes that are required for helicase activity. Accordingly, mutations in motif III can separate NTP hydrolysis from the unwinding activity of DEAD-box and DEAH-box proteins95,96. Motif VI is believed to participate in ATP binding, and mutations therein affect ATP hydrolysis. The structures of RNA helicases indicate that the remaining motifs (Ia, Ib, IV and V) are probably involved in RNA binding, although biochemical data are still lacking.

SKI2 FAMILY

RNA helicases that are related to DEAD-box proteins and named after Ski2, a yeast protein that is involved in RNA turnover. WALKER A AND B MOTIFS

Amino-acid consensus sequences that are present in nucleotide-triphosphate (NTP)binding proteins named after J. E. Walker who first described these motifs.

(Asp-Glu-Ala-Asp, or DEAD in one-letter code) gave the protein family its name. Recently, a further amino-terminal motif has been identified and named the Q-motif, which refers to a highly conserved glutamine residue in this motif3,4. All motifs are conserved at similar positions in the members of the protein family, defining a highly conserved core element that is flanked by divergent amino- and carboxy-terminal sequences. Today, over 500 different DEAD-box-protein sequences are present in protein databases (such as SwissProt and TrEMBL, see REF. 5) and despite this large number of proteins of various origins, the consensus sequences have remained essentially unchanged (BOX 1). Considering these conserved motifs, DEAD-box proteins can be clearly distinguished from RNA helicases of related families, such as the DEAH-box or the SKI2 families6. The alignments of members of these other families also show highly conserved motifs. Some of them are directly comparable to the motifs in the DEAD-box family, but others are not. Motif I, also known as the WALKER A MOTIF, and motif II, which corresponds to the Walker B motif, are present in all the families, and are recognizable as such. However, the striking difference in the DEAHbox and Ski2 families is the presence of a histidine residue in place of the second aspartic acid in motif II.

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Therefore, the RNA helicase families are often referred to as DExD/H proteins. Other motifs, such as motifs Ia or Ib, are more divergent. Significantly, so far, no helicase has been described that contains a mixture of motifs from different families. For example, the DEAD motif is always found together with the HRxGRxGR motif (motif VI), whereas DEAH is always associated with QRxGRxGR in the DEAH-box family. The newly discovered Q-motif and a conserved upstream aromatic residue are also highly characteristic of DEAD-box proteins. This motif was proposed to function as a sensor to determine the state of the bound ATP3,4. It is interesting that the use of NTPs is different between DEAD-box and DEAH-box families: whereas DEAD-box proteins require ATP, DEAH-box proteins are more promiscuous in their use of NTPs (for example, see REFS 4,7,8). However, even if the families are clearly distinct, it can be assumed, on the basis of the similarity between RNA and even DNA helicase structures, that the structures of the helicase cores in all the families are similar. Moreover, the respective motifs, as defined by the alignment of the members of each family, probably share their functional roles. It is intriguing that the families are so clearly distinct, as this indicates that there must be some fundamental differences between these families.

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Domain 1

ATPbinding cleft

the M. jannaschii DEAD-box protein is a rather compact molecule, its structure has a rotation of the domains relative to helicases from other families. A different relative orientation of the domains is also seen in the crystal structure of full-length eIF4A. It assumes a more ‘open’ or ‘more extended’ structure in which the amino- and carboxy-terminal domains are connected by an extended linker. This might be the consequence of the lack of substrates in these crystals.

Domain 2

VI

Q

I III

Cellular functions of DEAD-box proteins

V II Ia

IV

Ib

Figure 1 | Structure of the Methanococcus jannaschii DEAD-box protein. The Methanococcus jannaschii DEAD-box protein is similar in size to eIF4A. The conserved motifs16 are colour coded as in BOX 1. Clearly visible are the two domains 1 and 2, which form the ATP-binding cleft. All motifs occur at the surface of the two domains, consistent with them being involved in ATP binding (motifs Q, I, II and VI) and RNA binding (motifs Ia, Ib and V). The 3D reconstruction was created with Swiss-PDBviewer (see online links box) using the coordinates deposited at Protein Data Bank (1HV8).

In this review, we will discuss only DEAD-box proteins, except when otherwise stated. Because a large amount of functional data are from yeast, we will refer tacitly to this organism but refer explicitly to other organisms when needed. Structure–function relationship of RNA helicases

ARGININE FINGER

A catalytic residue that was first defined for Ras-GTPaseactivating proteins (RasGAPs), and that supplies a catalytic arginine residue into the active site of Ras to increase the reaction rate. SMALL NUCLEAR RNA (snRNA)

A small RNA molecule that functions in the nucleus by guiding the assembly of macromolecular complexes on the target RNA to allow site-specific modifications or processing reactions to occur. RNPase

An enzyme that causes the dissociation of a ribonucleoprotein (RNP) complex by disrupting protein–RNA interactions.

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Structural data for several RNA helicases have become available in recent years, providing a much better insight into the structure–function relationship of these proteins. Several structures of DNA helicases have been determined9,10, followed by the structure of the RNA helicase from hepatitis C virus, the DExH-box protein NS3 (REFS 11–13). More recently, the structures of two DEAD-box proteins, yeast eIF4A (REFS 14,15) and the Methanococcus jannaschii DEAD-box protein16, and of the amino-terminal half of the Bacillus stearothermophilus DEAD-box protein17, have been determined. Most importantly, despite the very low overall sequence conservation, the structural comparison of these different helicases showed that the catalytic helicase cores are largely superimposable. Together with genetic and biochemical data, the structural data have helped to clarify the role of the motifs (reviewed in REFS 18–20; FIG. 1). The M. jannaschii DEAD-box protein and eIF4A are among the shortest proteins in the DEAD-box family; consequently, their structures represent what is essentially a helicase core. Both structures showed that the conserved helicase core consists of two discrete domains (FIG. 1) that form a cleft in which ATP can bind and that are connected by a linker region (for a review, see REF. 18). In accordance with biochemical data, the Q-motif, motifs I and II in domain 1 and motif VI in domain 2, form part of the ATP-binding site between these domains. The second arginine residue in motif VI (HRxGRxGR) might perform a similar function as the ARGININE FINGER in G-proteins by sensing the γ-phosphate of the ATP18. Although


Many processes have been shown to require DEAD-box proteins. Because it is impossible to refer to all the published reports, we will discuss one or a few examples of every process (FIG. 2). As it can be seen from the following descriptions, the DEAD-box proteins are involved in very different processes but most of them probably have similar enzymatic activities. Indeed, in all these processes inappropriate or transitory RNA–RNA or RNA–protein interactions can be deleterious for subsequent steps, and therefore they require energy-driven motors to allow the faithful assembly of the complexes to accomplish forthcoming events. Transcription. Several DEAD-box proteins have been found to interact physically with the transcription machinery, or to colocalize with it 21–23. However, a clear function of an enzymatic activity of the DEAD-box proteins at the level of transcription has not yet been shown, and in many instances the enzymatic activity of the DEAD-box protein is not required for its function in transcriptional regulation21,22. An intriguing possibility for their presence at this stage is a connection between transcription and the later stages of RNA metabolism (see below). Pre-mRNA splicing. Newly transcribed pre-mRNA is modified at its 5′ and 3′ ends, and introns are spliced to create a mature messenger RNA for translation by the ribosome. Although the trans-esterification reactions in the splicing process are energetically neutral, it has been known for a long time that splicing requires energy from nucleotide-triphosphate (NTP) hydrolysis. A substantial part of the energy consumption is used for structural rearrangements in the stepwise assembly of the spliceosome, which involves five SMALL NUCLEAR RNAS (snRNAs) and over 70 proteins, including eight RNA helicases (BOX 2). Although the exact role of the different RNA helicases in spliceosome assembly is not known, it is generally believed that they are required for the unwinding of short RNA–RNA duplexes that are formed between the different snRNAs or pre-mRNA molecules. Recently, genetic evidence has indicated that the Sub2 and Prp28 proteins might also be involved in the dissociation of RNA–protein complexes24,25. Such an RNPase activity was shown in vitro for the viral DExH-box helicase NPH-II, and might be a property of other related RNA helicases26. Ribosome biogenesis. Similar events take place in the equally complex and dynamic process of ribosome biogenesis. During ribosome biogenesis, three out of the



REVIEWS

Nucleolus

Pre-60S

N Gp pp

Ribosome biogenesis

Polypeptide

Translation initiation

AAA AA

Pre-40S RNA polymerase

AAA AA

mRNA export AAA AA

G ppp

Mitochondria/chloroplasts

Pre-mRNA splicing G ppp

Gene expression

Nucleus Gp pp

Cytoplasm

RNA decay

AAA AA

Figure 2 | Cellular processes that require DEAD-box RNA helicases. Genetic and biochemical experiments in Saccharomyces cerevisiae, as well as in other organisms, have indicated several cellular functions for the DEAD-box proteins. These include ribosome biogenesis (for example, Fal1 (also known as eIF4AIII), Rok1, Spb4 and Dbp10), pre-mRNA splicing (for example, Prp28, Prp5 and Sub2), mRNA export (for example, Dbp5 and Sub2), translation initiation (for example, eIF4A, Ded1 and Vasa), organellar gene expression (for example, Mss116, Mrh4 and Cyt-19), and RNA decay (for example, RhlB, Dbp2 and Dhh1 (also known as Xp54)). It is thought that some DEAD-box proteins might function in two or more processes. In doing so, they probably serve as links or control elements for those cellular processes.

PSEUDO-URIDYLATION

The conversion of a uridine residue within an RNA chain into a pseudouridine residue, which requires the scission and reattachment of the base to the sugar. SMALL NUCLEOLAR RNA (snoRNA)

A small RNA molecule that functions in ribosome biogenesis in the nucleolus by guiding the assembly of macromolecular complexes on the target RNA to allow sitespecific modifications or processing reactions to occur. GROUP I INTRONS

A class of self-splicing introns, the excision of which in vivo is assisted by trans-acting protein factors. KINETICALLY TRAPPED

A particular substrate conformation that cannot be used further without it being refolded with external assistence. GUIDE RNA (gRNA)

A small RNA molecule that is complementary to a sequence that is to be modified or processed, and that guides proteins to this target.

of the nuclear pore30–32. Interestingly, it also interacts genetically and physically with the transcription machinery33,34. This indicates that Dbp5 is loaded onto newly synthesized mRNA and travels with it to the nuclear pore. This could serve as an integrated control for proper delivery of RNA polymerase II transcripts to the translation machinery and could contribute to the directionality of the transport. DEAD-box proteins that are required for RNA export might dissociate nuclear factors from the mRNA, allow the passage of mRNA through the pore, or prepare mRNA for the first round of translation.

four ribosomal RNA molecules are produced from a single large rRNA precursor. They are associated stepwise with ribosomal proteins involving over 100 trans-acting factors. Moreover, the 18S and 25/28S rRNAs are 2′-Oribose methylated and PSEUDO-URIDYLATED at many positions. These sequence-specific modifications are guided by SMALL NUCLEOLAR RNAS (snoRNAs)27,28 that are associated with trans-acting factors and must be added and removed from the pre-ribosomal particles during biogenesis to allow mutually exclusive interactions to occur. In yeast, 15 DEAD-box proteins (out of 25) and three RNA helicases from other families are required for ribosome biogenesis and most of them have homologous counterparts in other eukaryotes. As for pre-mRNA splicing, it has been suggested that RNA helicases in ribosome biogenesis are required for the unwinding of short duplexes of snoRNA–rRNA or rRNA–rRNA interactions. It is probable that some of them are required for the dissociation of RNA–protein interactions. Several bacterial DEAD-box proteins have also been shown to participate in ribosome biogenesis, but in contrast to most eukaryotic DEAD-box proteins, they do not seem to be essential under normal laboratory growth conditions, although they might be required at low temperatures29 (see below). RNA transport. Compartmentalization in eukaryotic cells requires the controlled transport of RNA molecules, proteins and complexes from the nucleus to the cytoplasm, and vice versa. The DEAD-box protein Dbp5 is required for the export of mRNA from the nucleus. It is found at the nuclear rim and it interacts with the cytoplasmic fibrils


Translation initiation. DEAD-box proteins have been shown to be required for translation initiation. On the basis of a large amount of experimental data, we can assume that eIF4A, which forms part of the cap-binding complex, unwinds or rearranges RNA-duplex structures at the 5′ end of eukaryotic mRNA to prepare it for scanning by the small ribosomal subunit.Another possibility is that eIF4A removes proteins from mRNA, which is probably coated with many proteins after exiting the nucleus35,36. Whereas several roles for eIF4A have been suggested, the function of another DEAD-box protein, Ded1, is not known, although its role in translation initiation has been indicated by several independent genetic and biochemical experiments37–40. The activities of eIF4A and Ded1 proteins might be required to various extents on different mRNA molecules41. This could mean that the helicase activities of these proteins fulfil regulatory roles in gene expression. In higher eukaryotes that undergo embryonic development, another DEAD-box protein, Vasa, is required for translational activation of germline-specific mRNAs in a spatially controlled fashion42. Although helicase activity is essential for the function of Vasa, its precise role in activating these messages is not clear 43. Organelle gene expression. DEAD-box proteins are also required for the expression of mitochondrial genomes in fungi (for example, Mss116, Mrh4, Cyt-19; REFS 44–46) and in higher eukaryotes (for example, MDX28; REF. 47). The Neurospora crassa Cyt-19 RNA helicase is required for the splicing of GROUP I INTRONS and it was suggested that it prevents the formation of KINETICALLY TRAPPED RNA intermediates. Interestingly, N. crassa has two genes that are highly related to yeast Mss116; one encoding an Mss116 homologue and the second, the Cyt-19 protein. This would indicate that Mss116 from yeast is also involved in the splicing of mitochondrial introns. However, if so, Mss116 must have another function in mitochondrial gene expression as it was shown to be required for mitochondrial function in a strain that is devoid of mitochondrial introns44. Yet another mitochondrial helicase, mHel61, is required for efficient editing of mitochondrial mRNAs in Trypanosoma brucei 48. Again, as in other modification processes, GUIDE RNAs (gRNAs) in large RNP complexes are required for the editing process and are probably targets for DEAD-box proteins. RNA decay. Finally, DEAD-box proteins are also required for the degradation of RNA molecules. The best studied case is the Escherichia coli RhlB protein that

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Box 2 | The dynamic spliceosome as a paradigm for the RNA helicase requirement The stepwise and dynamic assembly of the spliceosome is Exon 2 Exon1 U1 Intron a paradigm for the requirement of DEAD-box proteins in * Branchpoint cellular processes. The mosaic structure of many preU2 messenger RNAs requires the faithful excision of nonSub2 coding intron sequences. These sequences are defined by Prp5 simple 5′ exon–intron and 3′ intron–exon boundaries, U1 U2 * and they require specific internal intron sequences for splicing to occur. In addition to these cis-acting sequences, U4 U6 U1 U5 the splicing reaction requires a multitude of trans-acting Prp28 factors. These factors help to structure the pre-mRNA in a U4 U6 U2 * spatial fashion to permit intron excision to occur. Among U5 the trans-acting factors are five small nuclear Brr2 ribonucleoprotein (snRNP) complexes (U1, U2, U4, U5 U4 and U6) and several other protein factors, including U6 U2 * several RNA helicases from different families. The early U5 assembly of the spliceosome (that is, the assembly of the Prp2 pre-mRNA with snRNP U1) is an ATP-independent step. First Lariat The addition of snRNP U2, however, requires the input of trans-esterification U6 energy in the form of ATP. This step might be facilitated U5 * U2 by the Prp5 helicase, as this protein was shown to be preferentially stimulated by U2 snRNA. On the other Second Prp16 hand, the DECD-box helicase Sub2 (known in humans as trans-esterification U6 UAP56), which is otherwise a bona fide DEAD-box U5 * U2 protein, is also implied in this step. It has been suggested U6 U2 that Sub2 facilitates the removal of the Mud2 protein that U5 binds a pyrimidine-rich sequence close to the branch Prp22 Prp43 U6 point. Next, the tri-snRNP complex U4–U6–U5 joins the U5 * nascent spliceosome and displaces U1. This step, which U2 * involves the remodelling of several RNA–RNA and RNA–protein interactions, also requires ATP. On the basis of genetic experiments that involve strengthening or weakening the snRNP U1-pre-mRNA interaction, it was suggested that Prp28p is a candidate protein for this ATP-dependent step. The removal of snRNP U4 from the spliceosome requires the Ski2-family protein Brr2. This protein, which is part of the U5 snRNP, has been shown to exert helicase activity in an in vitro assay system97. It is at this stage that the first trans-esterification reaction occurs, which requires the DEAH-box protein Prp2. The subsequent steps (that is, the second trans-esterification reaction, the release of the mature mRNA and the recycling of the spliceosomal components) require other DEAH-box proteins, Prp16, Prp22 and Prp43, respectively. DEAD-box helicases are indicated in bold.

RNase E

An exonucleolytic RNase that digests RNA in the 3′→5′ direction POLYNUCLEOTIDEPHOSPHORYLASE

(PNPase). An enzyme that hydrolyzes single-stranded polyribonucleotides processively in the 3′→5′ direction. DEGRADOSOME

A complex of RNase E and polynucleotidephosphorylase (PNPase) that degrades RNA. The degradosome is assisted by a DEAD-box RNA helicase to allow degradation of structured RNAs EXOSOME

A complex of several exonucleases that, assisted by RNA helicases, degrades RNAs in the nucleus and the cytoplasm.

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forms a complex with RNase E and POLYNUCLEOTIDEPHOSPHO49 RYLASE (PNPase) . A minimal DEGRADOSOME that consists of RNase E, PNPase and the RhlB helicase has been shown to have ATP-dependent activity50. On the basis of the inability of RNAse E and PNPase to degrade structured RNA molecules, it has been suggested that RhlB is required for unwinding the substrate to allow degradation to occur. A similar model has been proposed for the eukaryotic EXOSOME, a multi-RNase complex, which is assisted in its activity by two DExH proteins, Ski2 and Dob1 (also known as Mtr4; REFS 51,52). Another DEADbox protein, Dhh1 (also known as Xp54), has recently been shown to be required for decapping and mRNA degradation in eukaryotes53. Interestingly, this protein was first variously described as being a part of storage particles for maternal mRNAs in Xenopus laevis 54,55, as part of the masking complex of maternal mRNA molecules in early development of clam oocytes56, and as being induced in certain colon cancers57. At present, the relationship between decapping, mRNA storage and overexpression in cancer is not clear. Nevertheless, it


has been shown that the X. laevis p54 protein can functionally substitute for the yeast Dhh1 protein, which indicates a close functional relationship throughout evolution58. Several roles for individual DEAD-box proteins? So far, DEAD-box proteins have generally been ascribed to specific processes. However, the new emerging concept of interlinkage of different processes through common components59 also applies to this family of proteins. A recent example is the linkage of splicing and mRNA export, as described for Sub2 (known as UAP56 in higher eukaryotes) by several groups60–62. Although conceptually it seems logical that unspliced pre-mRNA molecules should not be exported, it was a surprise that mutations affecting the splicing factor Sub2 also affect export of unspliced mRNA molecules, thereby clearly demonstrating the dual role of the Sub2 protein. Other DEAD-box proteins with dual roles are those that are involved in transcriptional regulation. Indeed, it has been shown for DP103 (also known as Gemin3) — a



REVIEWS mammalian protein that interacts with the SURVIVAL OF MOTOR NEURONS (SMN) PROTEIN that is involved in RNA splicing63 — that the carboxy-terminal flanking sequence is necessary and sufficient for transcriptional repression through its interaction with transcription factors. Whereas the helicase domain is not required for transcriptional repression, the carboxy-terminal sequence is required for helicase activity of the full-length protein. Finally, it is intriguing that the Ded1 helicase has been found by different approaches to be linked to pre-mRNA splicing and translation initiation, two processes that occur in two different cellular compartments. It is probable that other DEAD-box proteins will be required for more than one cellular process. Temporal and spatial specificity

SURVIVAL OF MOTOR NEURONS (SMN) PROTEIN

Product of the spinal muscular atrophy (SMA) disease gene. SMN is present in a large complex that functions in snRNP assembly, pre-mRNA splicing and transcription. MICHAELIS CONSTANT

(Km). The Michaelis constant is equal to the substrate concentration at which the reaction rate is half its maximal value. A high Km indicates a weak binding, and a low Km indicates strong binding. SERYL tRNA SYNTHETASE

An enzyme that catalyses the attachment of serine to the 3′ end of tRNAs containing the anticodons that correspond to serine. They belong to the family of aminoacyl-tRNA synthetases. TURNOVER NUMBERS

(kcat). The number of substrate molecules that are converted into product by an enzyme molecule in a unit of time when the enzyme is fully saturated with substrate. ∆G

The difference in free energy of a system at constant pressure and temperature. The more negative the ∆G, the more stable the duplex.

What keeps the RNA helicases from unwinding duplexes that should not be unwound, or from unwinding duplexes at inapproprate times, and what keeps them from interfering with unrelated processes? Indeed, despite the conservation in the core region, which comprises ~400 amino acids, most of the yeast DEAD-box proteins are essential and cannot be complemented by other members of the family, even when they are overexpressed (for an example, see REF. 64; for a review, see REF. 6). So, it is clear that elements other than the helicase core are required for the proper temporal and spatial regulation of these proteins. When compared with the other helicase families, which are relatively uniform in respect to size, DEAD-box and the related DEAH-box proteins range in size from ~400 to more than 1200 amino acids18,65, and are associated with a broad spectrum of cellular activities. In general, RNA helicases show very little specificity for RNA when they are tested in vitro (for example, see REFS 66–68). Moreover, none of the substrate–protein interactions that were observed in the crystal structure of the related DExH-box protein NS3 from hepatitis C virus involve the recognition of specific bases by the helicase core12. This might represent a general feature of these helicases because any sequence specificity might hinder the movement of the helicase along the nucleicacid strand (see below). From these observations, one could assume that substrate specificity might reside in the domains that flank the helicase core or in its interactions with other factors. In the case of the bacterial DEAD-box helicase DbpA69, the carboxy-terminal domain binds tightly to hairpin 92 of 23S rRNA, thereby conferring sequence specificity. The core helicase domain is believed to interact with a nearby single-stranded region of the RNA in a nonspecific fashion70. Recently, this fact has been exploited to show the transfer of specificity from one protein to another. The carboxy-terminal domain from a Bacillus subtilis homologue of DbpA, YxiN, was transferred to another bacterial DEAD-box protein from E. coli, SrmB — which by itself does not have any RNA specificity. In vitro, this new hybrid protein acquired the specificity of a DbpA-like protein71. Compared with prokaryotic enzymes such as DbpA, which shows sequence specificity in cis, so far, eukaryotic DEAD-box proteins lack a clear sequence or structure


specificity, except Prp5, which is weakly stimulated in its ATPase activity by the U2 snRNA72. This could indicate that eukaryotic DEAD-box proteins rely on their association with large RNP complexes to obtain substrate specificity73. Such interactions are important for optimal enzymatic activity (see below) and prohibit erroneous actions on unrelated substrates. In vitro activities of DEAD-box proteins

On the basis of the presence of helicase motifs and early biochemical assays with eIF4A and p68, all members of the DEAD-box family are considered to be helicases67,74,75. Although helicase activity is in accordance with the processes these proteins are involved in, for many proteins such an activity could not be shown in vitro. There are obvious reasons why the activity in vivo is not reflected by the in vitro ATPase and helicase properties of DEADbox proteins. For example, differences in substrates and lack of specific RNAs or protein cofactors lead to difficulties in the interpretation of biochemical data. Whereas helicase activity is often difficult to demonstrate, in vitro ATPase activity has been shown for many DEAD-box proteins. In most cases, ATP hydrolysis is RNA dependent, which clearly puts the biological function of DEAD-box proteins in the context of RNA. Affinities of DEAD-box proteins for ATP (expressed as the MICHAELIS CONSTANT (Km)), as determined in ATPase assays, range from 50 to 500 µM (for example, for eIF4A; REFS 70,76–78). These values do not denote particularly tight ATP binding compared with other proteins that bind both ATP and RNA (for example, 25µM for SERYL-tRNA SYNTHETASE79) but are well below the intracellular ATP concentrations (5–10 mM). The specific activities that have been measured for ATP hydrolysis are expressed as TURNOVER NUMBERS (kcat) and range from 3 min–1 for mammalian eIF4A (REFS 77,78) to 600 min–1 for DbpA70, which indicate large variations in vitro. In comparison, kcat for the viral DExH-box RNA helicase NS3 is ~2,000 min–1 (REF. 80). The weak in vitro ATPase activity of several DEAD-box proteins might reflect low intrinsic catalytic activity, the absence of cofactors, or the lack of proper post-translational modifications when they are expressed in heterologous systems. Also, weak stimulation by nonspecific RNA substrates might contribute to the low activity. Indeed, as was previously discussed, a specific hairpin in the 23S rRNA has been identified as a highly stimulating substrate for bacterial DbpA proteins70,81. The in vitro unwinding activity is generally tested using a partially double-stranded RNA (for example, REFS 66–68,75,82). In such assays, the nature and the length of the double-stranded region might have a significant influence on the ability of the helicase to successfully unwind. Furthermore, some of the putative helicases probably require protein cofactors that, for example, increase their affinity for the substrate73. Examples include mammalian and yeast Dbp5 proteins30,31. Using duplexes of a certain stability (where –1 ∆G = –35 to –44 kcal mol ) as substrates, Dbp5 is inactive in helicase assays in vitro, except when isolated from cell extracts by immunoprecipitation. This indicates that

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a On and off Pi

b Translocation Pi

c Translocation and unwinding Pi

ATP ADP+Pi

Figure 3 | Possible modes of unwinding by DEAD-box RNA helicases. ATP-dependent unwinding can be envisaged in several ways. Here we describe three distinct modes of unwinding, although there might be intermediate mechanisms. a | The first model depicts a DEAD-box RNA helicase that associates and dissociates from a single-stranded RNA tail and might thereby destabilize a duplex. ATP hydrolysis is required to release the RNA. b | In the second model, the helicase is walking actively along the single-stranded RNA, and once it encounters the duplex, again, it destabilizes a breathing molecule. c | In the third model, the helicase uses energy from ATP hydrolysis for translocation and active unwinding. For simplicity, the enzyme is represented as a monomer with an ATP (red) or ADP (blue) in the cleft formed by domains 1 and 2. Pi, inorganic phosphate.

other factors are required for the unwinding of such duplexes. Another example is eIF4A, the helicase activity of which was originally shown to be dependent on the RNA-binding protein eIF4B (REF. 75). Later, Rogers et al., using a population of relatively unstable duplexes83 (where ∆G = –16 to –25 kcal mol–1; by contrast, in those previously used, ∆G = –35 kcal mol–1; REF. 75), were able to show helicase activity for eIF4A in the absence of eIF4B. This indicated that this activity is sensitive to the difference in the stability of the duplexes used. The authors suggested that eIF4A might function to partially unwind duplexes; according to the stability of the remaining duplex, either melting would occur or release of eIF4A would allow re-annealing. Therefore, eIF4A is, at least in vitro, a non-processive helicase, which destabilizes the duplexes by preventing their re-annealing, rather than by actively displacing strands. This might be valid for many DEAD-box helicases. It is important to realize that naturally occurring duplex RNAs that are present in the described processes are, in general, fairly short (for example, see REFS 28,84). Helicases are known to require a loading strand and most of them show a directional activity; that is, either a 5′→3′ or a 3′→5′ activity 85. It was therefore surprising that experiments with eIF4A and p68 showed that they had bidirectional activity 67,75,86. Such a bidirectional activity indicates that the RNA helicase does not use the loading strand as a guiding track, but rather that the unwinding operates through a continuous ‘on and off ’ movement from the substrate (see below). Importantly, the activity might become unidirectional if provided with a specific substrate, as exemplified by DbpA70, or by an accessory protein clamping the helicase to the substrate.

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Although the term helicase implies the unwinding of a duplex, the function of RNA helicases might also involve the dissociation of an RNA–protein complex (RNPase activity). The protein would bind to a singlestranded RNA molecule and translocate along RNA until it encounters a bound protein. The encountering event would then be sufficient to weaken the RNA–protein interaction and eventually lead to the dissociation of the protein–RNA complex26. Such an activity would make sense in many processes, such as splicing, RNA export, ribosome biogenesis and translation. So, it is probable that all DEAD-box proteins have ATPase and displacing activity in vivo, but that these activities are not always manifested in an in vitro context. In particular, the in vivo activity is influenced by the substrate and interacting partner proteins that contribute to the spatial and temporal control of these enzymes (see above). How do DEAD-box proteins exert their function?

As expected from helicases, DEAD-box proteins rearrange duplexes in an ATP-dependent manner. This activity can be envisaged in several ways and we present here three non-exclusive views (FIG. 3). In a first model, the DEAD-box protein functions simply by an ‘on–off’ mechanism, which implies ‘breathing’ (that is, thermal denaturation of the duplex ends is used by the protein to attach to the open end). Such thermal breathing of a duplex has been estimated to occur at a rate of about 1,000 times per second (REF. 19,87). Once the protein is bound, the duplex is in an unstable state, which can result either in complete thermal melting or in dissociation of the protein from the RNA without unwinding. In this ‘on–off’ type of model, ATP hydrolysis is used to release the substrate from the protein to enable recycling, which is necessary for a new round of binding. A possible example of this mechanism could be, at least in vitro, eIF4A, which has very low helicase activity by itself. This is consistent with the requirement for a high excess of protein (200-fold excess of protein over substrate, see REFS 88,35) and the reported unwinding of blunt-ended substrates. Biochemical data that show a decrease in RNA affinity upon ATP hydrolysis support such a scenario77,78. In a model of more efficient enzymes, DEAD-box proteins use the energy that is gained from ATP hydrolysis to translocate along single-stranded RNA. Once the protein encounters the duplex, it takes advantage of thermal melting to proceed along the transiently denatured strand to prevent re-annealing. In this model of passive unwinding, ATP hydrolysis is required for translocation to enhance the chance of denaturation by reducing the rate of re-annealing. Further thermal melting and translocation will finally result in dissociation of the duplex. Depending on the affinity of the helicase for the substrate, the activity can be more or less processive. Such a mode of action requires less protein than the first model and could possibly apply to DEAD-box proteins, such as Ded1, that, in contrast to eIF4A, can unwind a large excess of substrate in an ATP-dependent fashion (O. Cordin, J. Banroques, N.K. Tanner and P.L., unpublished observations).



REVIEWS

Archaea Sulfolobus (1) Methanococcus (1)

Chlamydia (0)

Pyrococcus (0) Escherichia (5) Halobacterium (0) Bacillus (5) Eubacteria

Thermotoga (0)

Homo sapiens (38) Saccharomyces (25) Arabidopsis (55) Eukaryotes

Figure 4 | Phylogram of DEAD-box proteins from the three kingdoms of life. Representative examples of DEADbox proteins from the three kingdoms were used to show the different numbers of DEAD-box proteins that are encoded in genomes that have been fully sequenced. The phylogram was constructed with the ClustalW program (default settings) using the 16S/18S rRNA from the indicated species. It does not present a fully calculated phylogenetic tree, but rather a tool to represent the three kingdoms. The numbers for Homo sapiens, Arabidopsis thaliana and Saccharomyces cerevisiae are from published reports6,98,99. The numbers for the remaining organisms were obtained by BLAST searches using the yeast eIF4A sequence against translated sequences or against the DNA sequences of fully sequenced genomes (see BLAST searches for putative helicases in online links box).

In a third model, active destabilization of an RNA duplex requires the energy from ATP to power both translocation and active disruption of the base pairs at the junction of single-stranded and doublestranded RNA. This last model applies more to processive DNA helicases (reviewed in REF. 19) but could also be used by RNA helicases89. In our present view of the in vivo activity of DEAD-box proteins, it is not likely that these proteins require a processive helicase activity, as most, if not all, of the potential substrates of DEAD-box proteins represent only short duplexes as, for example, in the case of rRNA–snoRNA interactions90. In principle, all three models can also apply to the activity of RNPases, although, intuitively, more proficient enzymes such as those presented in the second and third models are more likely to carry out such a function. Are DEAD-box proteins ubiquitous? MICROSPORIDIA

Obligate intracellular parasites that have a reduced genome content. NUCLEOMORPH

A vestigial nucleus-like structure that harbours the smallest eukaryotic genomes of an algal endosymbiont.

RNA metabolism is an evolutionarily very old process and is present in all unicellular and multicellular organisms. It could therefore be expected that DEAD-box proteins are present in all organisms. However, a search for such proteins in sequence databases (SwissProt/TrEMBL, NCBI genome sequences) reveals interesting differences between species (FIG. 4). At first glance, it is obvious that eukaryotic cells have many more DEAD-box proteins


than bacteria and archaea. Moreover, many, if not all, DEAD-box proteins in E. coli are dispensable for growth under laboratory growth conditions, whereas, in yeast, 17 out of 25 DEAD-box proteins are essential6,65. Nevertheless, it can be argued that bacterial RNA helicases are necessary under certain growth conditions, such as low temperature. Indeed, many bacteria (for example, Anabaena variabilis) and archaea (for example, Methanococcoides burtonii) induce the synthesis of DEAD-box proteins specifically under cold-shock conditions91,92. In line with this argument, obligate intracellular pathogens of the species Chlamydia do not possess DEAD-box proteins, presumably because they always grow at the temperature of their hosts. However, other eubacteria or archaebacteria (for example, Borrelia burgdorferi or Aeropyrum pernix and Methanopyrus kandleri) do not seem to encode DEAD-box proteins either. It is noticeable that the absence of genes that encode DEAD-box proteins is not related to the number of genes that is present in an organism, as rudimentary eukaryotes such as the MICROSPORIDIA Encephalitozoon cuniculi and NUCLEOMORPH Guillardia theta encode 11 and 5 DEAD-box proteins, out of a total of 1,997 and 531 proteins, respectively. So, a minimal number of DEAD-box proteins seems to be necessary for eukaryotes, but not for eubacteria or archaea. In conclusion, DEAD-box proteins are involved in many processes and have important roles in RNA metabolism. This is also reflected by the fact that several DEAD-box proteins are overexpressed in cancer cells. Moreover, RNA helicases from related families are essential for the propagation of many viruses that cause human diseases. So, understanding the mechanism, structure and function of RNA helicases might help to design therapies against these diseases or viral infections. As an example, the characterization of two related RNA helicases, human eIF4A and hepatitis C virus NS3 (REF. 88), has provided leads for designing specific inhibitors that target the viral NS3 helicase. The identification of family-specific conserved features opens the possibility to develop antagonists against specific helicases or helicase families. Although recent years gave us plenty of exciting insights into the biology, structure and function of DEAD-box proteins, many essential questions remain to be answered. Among them are of course the identification of DEAD-box protein targets and the way they find their targets. What makes DEAD-box proteins specific for one particular step and what determines their function in these crowded environments? Clearly, biochemical approaches including crosslinking experiments and genetic screens, such as the isolation of bypass suppressors, will help to answer these questions. Following directly from the question about substrates is also the regulation of DEAD-box proteins — an RNA helicase should exert its activity solely on its substrate in the right place and at the right time. So, key challenges for the future are the elucidation of the exact mechanism of the ‘unwindase’ activity of these proteins and the temporal and spatial control of the enzymatic activity.

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Acknowledgements We are grateful to members of the laboratory and the helicase community for continuous discussion. We acknowledge highly stimulating discussions and pertinent comments about this manuscript from M. Altmann, D. Belin, O. Cordin, F. Fuller-Pace, T. Lacombe, G. Owttrim, N.K. Tanner and the three referees. Work in our laboratory is supported by the Swiss National Science Foundation and the state of Geneva.

Competing interests statement The authors declare that they have no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/entrez/ Cyt-19 | DbpA | mHel61 | Rhlb | SrmB | YxiN Flybase: http://flybase.bio.indiana.edu/ Vasa The Protein Data Bank: http://www.rcsb.org/pdb/ 1HV8 Saccharomyces genome dtabase: http://www.yeastgenome.org/ Brr2 | Dbp5 | Ded1 | Dhh1 | Dob1 | eIF4A | Mrh4 | Mss116 | Mud2 | Prp5 | Prp16 | Prp22 | Prp28 | Prp43 | Ski2 | Sub2 Swiss-Prot: http://us.expasy.org/sprot/ DP103 FURTHER INFORMATION RNA helicases: http://www.medecine.unige.ch/~linder/RNA_helicases.html Swiss-PDBviewer: http://www.expasy.org/spdbv/ BLAST searches for putative helicases: http://www.medecine.unige.ch/~linder/bacterial_helicases.html Access to this interactive links box is free online.

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