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RNA sequence‐ and shape‐dependent recognition by proteins in the ribonucleoprotein particle

RNA sequence‐ and shape‐dependent recognition by proteins in the ribonucleoprotein particle review review RNA sequence- and shape-dependent recognition by proteins in the ribonucleoprotein particle Richard Stefl, Lenka Skrisovska & Frédéric H.-T. Allain Swiss Federal Institute of Technology Zürich, Zürich, Switzerland At all stages of its life (from transcription to translation), an RNA recognize primarily the shape of the RNA or both the sequence and transcript interacts with many different RNA-binding proteins. The the shape. Other types of RNA-binding domains, such as the composition of this supramolecular assembly, known as a ribo- K-homology (KH) domain or the oligonucleotide/oligosaccharide- nucleoprotein particle, is diverse and highly dynamic. RNA-binding binding (OB) fold, have recently been reviewed and are not proteins control the generation, maturation and lifespan of the RNA discussed here (Messias & Sattler, 2004). transcript and thus regulate and influence the cellular function of the encoded gene. Here, we review our current understanding of RNA shape-dependent recognition by double-stranded RBM protein–RNA recognition mediated by the two most abundant RNA- The dsRBM is a 70–75 amino-acid domain with a conserved αβββα binding domains (the RNA-recognition motif and the double-stranded protein topology in which the two α-helices are packed along one RNA-binding motif) plus the zinc-finger motif, the most abundant face of a three-stranded anti-parallel β-sheet (Fig 1; Fierro-Monti & nucleic-acid-binding domain. In addition, we discuss how not only Mathews, 2000; St Johnston et al, 1992). These domains occur mostly the sequence but also the shape of the RNA are recognized by these in multiple copies (up to five) and have so far been found in 388 three classes of RNA-binding protein. eukaryotic proteins, 72 of which are human (data taken from the Keywords: double-stranded RNA-binding motif; RNA-binding SMART database; Letunic et al, 2004). These proteins have an essen- proteins; RNA recognition; RNA-recognition motif; zinc-finger motif tial role in RNA interference, RNA processing, RNA localization, EMBO reports (2005) 6, 33–38. doi:10.1038/sj.embor.7400325 RNA editing and translational repression (Doyle & Jantsch, 2002; Saunders & Barber, 2003). Introduction So far, only three structures of dsRBMs in complex with dsRNA The association of RNA-binding proteins (RBPs) with RNA tran- have been determined (Table 1): a 1.9 Å crystal structure of the sec- scripts begins during transcription. Some of these early-binding ond dsRBM of Xenopus laevis RNA-binding protein A (Xlrbpa2) RBPs remain bound to the RNA until it is degraded, whereas others bound to two coaxially stacked dsRNA molecules, each 10 bp long recognize and transiently bind to RNA at later stages for specific (Ryter & Schultz, 1998); a nuclear magnetic resonance (NMR) struc- processes such as splicing, processing, transport and localization ture of the third dsRBM from the Drosophila Staufen protein in com- (Dreyfuss et al, 2002). The RBPs cover the RNA transcripts and con- plex with a symmetrical GC-rich 12-bp duplex capped by a UUCG trol their fate. Some RBPs function as RNA chaperones (Lorsch, tetraloop (Ramos et al, 2000); and an NMR structure of the dsRBM of 2002) by helping the RNA, which is initially single-stranded, to form Rnt1p (an RNase III homologue from budding yeast) bound to a various secondary or tertiary structures. When folded, these struc- 14-bp RNA duplex capped by an AGAA tetraloop (Wu et al, 2004). tured RNAs, together with specific RNA sequences, act as a signal All three structures have several common features that reveal how a for other RBPs that mediate gene regulation. Here, we review our dsRBM is able to bind to any dsRNA but not to dsDNA, regardless of current structural understanding of protein–RNA recognition medi- its base composition. The dsRBMs interact along one face of the ated by the two most abundant RNA-binding domains, the RNA- RNA duplex through both α-helices and their β1–β2 loop (Fig 1). The recognition motif (RRM) and the double-stranded RNA-binding contacts with the RNA cover 15 bp that span two consecutive minor motif (dsRBM), and by the most abundant nucleic-acid-binding grooves separated by a major groove. In all three structures, the con- motif, the CCHH-type zinc-finger domain. We discuss how these tacts to the sugar-phosphate backbone of the major groove and of three small domains recognize RNA: some bind single-stranded one minor groove (Fig 1) are mediated by the β1–β2 loop and the RNA by direct readout of the primary sequence, whereas others amino-terminal part of α-helix 2. These interactions are non- sequence-specific as they involve 2’-hydroxyls and phosphate oxy- gens and are perfectly adapted to the shape of an RNA double helix. Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology Zürich, ETH-Hönggerberg, CH-8093 Zürich, Switzerland By contrast, the interactions mediated by α-helix 1 are different in all Corresponding author. Tel: +41 (0)1 63 33940; Fax: +41 (0)1 63 31294; three complexes. In the dsRBM of Xlrbpa2, α-helix 1 interacts non- E-mail: [email protected] specifically with the other minor groove of the RNA (Fig 1A), with Submitted 22 September 2004; accepted 26 November 2004 a few contacts to the bases. In the dsRBM of Staufen, α-helix 1 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 1 | 2005 33 © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review target but not its sequence. dsRBMs are highly conserved and have α-helix 2 the same structural framework, but are chemically distinct through variations in key residues. The structure of the dsRBM of Rnt1p in complex with RNA highlights the essential role of the α-helix 1 in α-helix 1 the recognition of structured elements that deviate from regular dsRNA. The α-helix 1 is the least-conserved secondary structure ele- β1–β2 loop ment among various dsRBMs and seems to have a different spatial arrangement relative to the rest of the domain in different dsRBMs. This variability may be an important factor as many biochemical experiments have shown that dsRBM-containing proteins have bind- ing specificity for a variety of RNA structures, such as stem–loops, internal loops, bulges or helices with mismatches (Doyle & Jantsch, 2002; Fierro-Monti & Mathews, 2000; Ohman et al, 2000; Stephens et al, 2004). Clearly, further structures are needed to decipher the extent of RNA shape-dependent recognition by dsRBMs. β-helix 3 RNA sequence- and shape-dependent recognition by an RRM The RRM is the most common RNA-binding motif. It is a small pro- α-helix 2 tein domain of 75–85 amino acids with a typical βαββαβ topology that forms a four-stranded β-sheet packed against two α-helices β1–β2 loop (Mattaj, 1993). RRMs are found in about 0.5%–1% of human genes α-helix 1 (Venter et al, 2001) and are often present in multiple copies (up to six per protein). RRM-domain-containing proteins are involved in many cellular functions, particularly messenger RNA and ribosomal RNA processing, splicing and translation regulation, RNA export and RNA stability (Dreyfuss et al, 2002). So far, ten structures of an RRM in complex with RNA have been determined using either NMR spectroscopy or X-ray crystallography (Table 1). These structures reveal the complexity of protein–RNA recognition mediated by the RRM, which often involves not only protein–RNA interactions but also RNA–RNA and protein–protein Fig 1 | Double-stranded RNA recognition by double-stranded RNA-binding interactions. All ten structures reveal some common features. The motifs. (A) The double-stranded RNA-binding motif (dsRBM) of Xlrbpa2 main protein surface of the RRM involved in the interaction with the bound to dsRNA (Ryter & Schultz, 1998). The α-helix 1 (in red), amino- RNA is the four-stranded β-sheet, which usually contacts two or three terminal part of α-helix 2, and β1–β2 loop recognize non-sequence- nucleotides (exemplified here by the RRM1 of sex-lethal; Fig 2A; specifically the shape of dsRNA. Backbones are coloured blue and light blue for Handa et al, 1999). The nucleotides are located on the surface of the two co-axially stacked duplexes. (B) The dsRBM of Rnt1p bound to the β-sheet, with the bases oriented parallel to the β-sheet plane and stem–loop closed by the AGNN tetraloop (backbone in yellow and bases in often packed against conserved hydrophobic side-chains (usually black; Wu et al, 2004). The α-helix 1, a key element for shape-specific aromatics). These two or three nucleotides are recognized sequence- recognition by dsRBMs, is highlighted in red. The dsRBM of Rnt1p has an specifically by interactions with the protein side-chains of the β-sheet additional carboxy-terminal α-helix 3 (in black), that modulates the and with the main-chain and side-chains of the residues carboxy- conformation of α-helix 1 (Leulliot et al, 2004). Side chains involved in terminal to the β-sheet. Interestingly, it seems that almost all possible intermolecular interactions are shown. sequences (doublets or triplets) can be accommodated on such a surface as the RNA sequences are different in each structure (Table 1). Often, RRM-containing proteins bind more than three interacts with a UUCG tetraloop that caps the RNA double helix. nucleotides and recognize longer single-stranded RNA (for exam- Although the UUCG tetraloop is not a natural substrate of Staufen, ple, poly(A)-binding protein (PAPB; Deo et al, 1999), sex-lethal this finding led to the proposal that α-helix 1 modulates the speci- (Handa et al, 1999), Hu protein D (HuD; Wang & Hall, 2001), ficity of individual dsRBMs (Ramos et al, 2000). Indeed, this was heterogeneous nuclear RNP A1 (hnRNP A1; Ding et al, 1999), recently confirmed by the structure of the dsRBM of Rnt1p bound to nucleolin (Allain et al, 2000; Johansson et al, 2004), RNA its natural RNA substrate (Fig 1B), in which α-helix 1 recognizes the stem–loops U1A (Oubridge et al, 1994), U2B’’ (Price et al, 1998), specific shape of the minor groove created by the conserved AGNN nucleolin (Allain et al, 2000)) or even internal loops (U1A; Allain –9 –1 tetraloop (Wu et al, 2004). The α-helix 1, the conformation of which et al, 1997; Varani et al, 2000), all with high affinity (K ≈ 10 M ). is stabilized by an additional carboxy-terminal α-helix 3 (Fig 1B; In U1A, U2B’’, nucleolin and sex-lethal, two loops between the Leulliot et al, 2004), is tightly inserted into the RNA minor groove secondary-structure elements of the RRM (the β2–β3 loop and and contacts the sugar-phosphate backbone and the two non- the β1–α1 loop) are essential for additional contacts with the RNA conserved tetraloop bases, whereas the conserved A and G bases are (Fig 2B). These loops vary significantly in size and amino-acid not involved in the interactions (Wu et al, 2004). This structure illus- sequence between the different RRMs. In the RRM of CBP20, the trates how this dsRBM recognizes the specific shape of its RNA C- and N-terminal extensions (which are stabilized by the cognate 34 EMBO reports VOL 6 | NO 1 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review Table 1 | Various structures of RNA-binding proteins bound to RNA Complex RNA secondary No. of RBDs in RNA sequence Function Reference; Protein Data Bank structure structures vs in recognized specifically (PDB) ID full-length protein by RRM β-sheet dsRBM type Second dsRBM Duplex 1/3 – hnRNP association, Ryter & Schultz, 1998; 1DI2 of Xlrbpa2 translation repression Third dsRBM Stem–loop 1/5 – mRNA localization, Ramos et al, 2000; 1EKZ of Staufen translation control dsRBM of Rnt1p Stem–loop 1/1 – RNA processing Wu et al, 2004; 1T4L RRM type N-terminal RRM Stem–loop 1/2 CAG Pre-mRNA splicing Price et al, 1998; 1A9N of U2B’’ (in U2B’’– U2A’–RNA complex) N-terminal RRM Stem–loop 1/2 CAC Pre-mRNA splicing Oubridge et al, 1994; 1URN of U1A N-terminal RRM Internal loop 1/2 CAC Pre-mRNA splicing Allain et al, 1997; 1AUD, of U1A Varani et al, 2000; 1DZ5 Two N-terminal Stem–loop 2/4 RRM1-CG Ribosome biogenesis Allain et al, 2000; 1FJE, RRMs of nucleolin RRM2-UC Johansson et al, 2004; 1RKJ Two N-terminal Single strand 2/4 RRM1-AAA Translation initiation Deo et al, 1999; 1CVJ RRM of PABP RRM2-AAA Two RRMs Single strand 2/2 RRM1-UUU Alternative splicing Handa et al, 1999; 1H2T of sex-lethal RRM2-UGU Two N-terminal Single strand 2/3 RRM1-UUU mRNA stability, Wang & Hall, 2001; RRMs of HuD RRM2-UU translation regulation 1FXL, 1G2E RRM of CBP20 Single strand 1/1 m7GpppG Maturation of pre-mRNA Mazza et al, 2002; 1H2V and U-rich snRNA Zinc finger Fourth, fifth and sixth Truncated 3/9 – Transcription regulation Lu et al, 2003; 1UN6 zinc fingers (CCHH- 5S RNA type) of TFIIIA First and second Single strand 2/2 – RNA processing Hudson et al, 2004; 1RGO zinc fingers (CCCH- and degradation type) of TIS11d a b c Two coaxially stacked dsRNA (each 10-bp long). 12-bp duplex capped by a non-physiologically relevant UUCG tetraloop. Consists of loop A, loop E and helices I, IV and V. CBP20, cap-binding protein 20; dsRBM, double-stranded RNA-binding motif; hnRNP, heterogeneous nuclear ribonucleoprotein; HuD, Hu protein D; PABP, poly(A)-binding protein; RBD, RNA-binding domain; Rnt1p, RNase III homologue; RRM, RNA-recognition motif; TFIIIA, transcription factor IIIA; Xlrbpa2, Xenopus laevis RNA-binding protein A. protein CBP80) provide a tight binding pocket for the 5’ capped RNA recognition by zinc fingers RNAs (7-methyl-G(5’)ppp(5’)N, where N is any nucleotide; Fig 2C; CCHH-type zinc-finger domains are the most common DNA-binding Mazza et al, 2002). In proteins that contain several RRMs, high- domain found in eukaryotic genomes. Typically, several fingers are affinity binding can only be achieved by the cooperative binding used in a modular fashion to achieve high sequence-specific recogni- of at least two RRMs to the RNA (for example, in nucleolin tion of DNA (Miller et al, 1985). Each finger displays a ββα protein fold 2+ (Fig 2D), PABP and sex-lethal). In addition to the β-sheet–RNA in which a β-hairpin and an α-helix are pinned together by a Zn ion. contacts, interactions between the inter-domain linker and the DNA-sequence-specific recognition is achieved by the interactions RNA and between the RRMs themselves contribute to the marked between protein side-chains of the α-helix (at position –1, 2, 3 and 6, increase in affinity compared with the binding of the individual for the canonical arrangement) and the DNA bases in the major domain alone. These structures show that the RRM is a platform groove (Fig 3A; Wolfe et al, 2000). However, there is increasing evi- with a large capacity for variation in order to achieve high RNA- dence that zinc fingers are also used to recognize RNA (Finerty & Bass, binding affinity and specificity. For example, it is remarkable that a 1997; Mendez-Vidal et al, 2002; Picard & Wegnez, 1979; Theunissen single domain like nucleolin RRM2 contacts only two nucleotides, et al, 1992). The crystal structure of three zinc fingers (fingers 4–6) of whereas U1A RRM1 contacts 12 nucleotides and the RRM of Y14 transcription factor IIIA (TFIIIA) in complex with a 61-nucleotide frag- (Fribourg et al, 2003) does not contact RNA but rather another pro- ment of the 5S RNA (Lu et al, 2003) provided the first insight into RNA tein. This fascinating plasticity of the RRM explains why it is so recognition by CCHH-type zinc fingers. In this structure, finger 4 binds abundant and why it is involved in so many different biological to loop E, finger 5 to helix V, and finger 6 to loop A (Fig 3B). Finger 4 functions; however, this plasticity makes it difficult to predict how recognizes loop E by specifically interacting with a bulged guanosine the RRM achieves RNA recognition. (Fig 3C) and, similarly, finger 6 recognizes loop A by specifically ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 1 | 2005 35 © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review A C N-terminal C-terminal N-terminal 3' m7GpppG 5' C-terminal B D C-terminal C-terminal Interdomain linker N-terminal β2–β3 loop N-terminal β1–α1 loop Fig 2 | RNA recognition by RNA-recognition motifs. The similarities and differences are highlighted in red and yellow, respectively. (A) RNA-recognition motif 1 (RRM1) of sex-lethal (shown as a ribbon model) interacts with the triplet UUU (shown as a stick model; Handa et al, 1999). The four-stranded β-sheet (in red) recognizes three nucleotides—this is the canonical mode of RRM–RNA interaction. (B) RRM1 of U1A bound to an RNA internal loop (Allain et al, 1997). The four-stranded β-sheet (in red) recognizes three nucleotides. The β2–β3 loop and the β1–α1 loop (in yellow) contact additional RNA residues. (C) RRM of CBP20 complexed with m GpppG (7-methyl-G(5’)ppp(5’)G; Mazza et al, 2002). C- and N-terminal extensions (in yellow) provide additional protein–RNA contacts that creates a specific binding pocket for 5’ capped RNAs. (D) RRMs 1 and 2 of nucleolin bound to an RNA stem–loop (Allain et al, 2000). The four-stranded β-sheet of RRM2 (in red) recognizes only two nucleotides. The interdomain linker (in yellow) participates in the recognition of the hairpin architecture. interacting with two bases (an adenine and a cytosine) that also bulge multiple contacts between basic amino acids of the α-helix and the out from the rest of the RNA (Fig 3B). The specific recognition of the RNA sugar-phosphate backbone (Fig 3D). RNA by both fingers 4 and 6 is achieved by side-chain contacts from In contrast to the above-mentioned CCHH zinc fingers, another the N-terminal parts of the α-helix (at position –1, 1 and 2; Fig 3C). The class of zinc fingers (CCCH-type) was recently found to adopt a dif- interaction of finger 5 with helix V differs from the ones made by fingers ferent fold and to recognize sequence-specifically single-stranded 4 and 6. In this case, finger 5 recognizes a short RNA double helix by RNA (Hudson et al, 2004). In this NMR structure, sequence-specific 36 EMBO reports VOL 6 | NO 1 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION © 2005 Nature Publishing Group Loop E Helix V RNA recognition by RBPs R. Stefl et al. review F6 AB C F5 Loop A Helix V F4 F4 Loop E F5 Fig 3 | DNA vs RNA recognition by CCHH-type zinc fingers. (A) Zinc finger 2 of Zif 268 bound to double-stranded DNA (Pavletich & Pabo, 1991). The α-helix of the zinc finger (in red) inserts into the DNA major groove; base contacts are made from positions –1, 2, 3 and 6 of the α-helix (the protein side-chains are shown as an element-type coloured stick model). The DNA bases that are recognized by the finger are coloured yellow. (B) Overall view of the complex of transcription factor IIIA (TFIIIA) fingers 4–6 (F4–F6) and 61-nucleotide 5S RNA (Lu et al, 2003). The protein and RNA are represented as ribbon models. The bulged bases involved in the recognitions are highlighted in yellow. Cyan balls represent zinc ions. (C) TFIIIA finger 4 (F4) bound to loop E (Lu et al, 2003). The α-helix (in red) of the finger 4 specifically interacts with a guanosine base that bulges out (in yellow); the base contacts are made from the side-chain at position –1, 1 and 2 of the α-helix. (D) TFIIIA finger 5 (F5) bound to helix V (Lu et al, 2003). The α-helix (in red) of finger 5 recognizes the dsRNA shape by non-sequence-specific contacts to the RNA sugar-phosphate backbone. RNA recognition is achieved by a network of intermolecular hydro- regulation. The enormous diversity of interactions observed in pro- gen bonds between the protein main-chain functional groups and tein–RNA complexes indicates that a simple recognition code is the Watson–Crick edges of the bases (Hudson et al, 2004). These unlikely to exist in the world of protein–RNA interactions. However, structures reveal that zinc fingers bind to RNA differently to the way two unifying themes may be inferred from the known complexes: the they do to DNA. The CCHH-type zinc fingers have two modes of recognition of the primary RNA sequence and/or the recognition of RNA binding. First, the zinc fingers interact non-specifically with the the RNA shape by individual RBPs. In a simplistic view, the RRMs, backbone of a double helix, and second, the zinc fingers specifically dsRBMs and CCHH-type zinc fingers seem to be shaped to recognize recognize individual bases that bulge out of a structurally rigid ele- single-stranded RNA, double-stranded RNA and RNA bulges, respec- ment. The CCCH-type zinc fingers show a third mode of RNA binding, tively. However, we have shown here by reviewing several recent in which the single-stranded RNA is recognized in a sequence-spe- protein–RNA complex structures that, the RRM and, to a lesser cific manner. Taken together, zinc fingers represent a unique class of extent, the dsRBMs and the CCHH-type zinc fingers have evolved to nucleic-acid-binding proteins that are capable of a direct readout recognize specifically a rich repertoire of RNAs in terms of length, of the DNA sequence within a DNA double helix, a direct readout of sequence and structure. This is achieved in three ways: first, by the the RNA sequence within single-stranded RNA, and an indirect subtle amino-acid change in variable regions of the domains, namely readout of the RNA as they recognize the shape of the RNA rather the β2–β3 and the β1–α1 loops in the RRM, α-helix 1 in the dsRBM than its sequence. Of course, more structures of CCHH-type and and the α-helix in the zinc fingers; second, by multiplication of the CCCH-type zinc fingers in complex with RNA will need to be domains to achieve higher affinity through cooperative binding; and determined to generalize their mode of RNA recognition. third, by extension of the protein domain. Although more structures still need to be determined, it might soon be possible to predict which Conclusions RBP binds to which RNA, and how it recognizes its target. As a conse- Proteins that contain RNA-binding domains and their interactions quence, post-transcriptional gene expression and its regulation could with RNA have important roles in all aspects of gene expression and be understood and controlled at the atomic level. ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 1 | 2005 37 © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review Ohman M, Kallman AM, Bass BL (2000) In vitro analysis of the binding of ACKNOWLEDGEMENTS ADAR2 to the pre-mRNA encoding the GluR-B R/G site. RNA 6: We apologize to authors whose work could not be cited due to space 687–697 constraints. The authors are supported by the Swiss National Science Oubridge C, Ito N, Evans PR, Teo CH, Nagai K (1994) Crystal structure at Foundation (No. 31-67098.01), the Roche Research Fund for Biology at the 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal ETH Zurich (F.H.-T.A.), and the European Molecular Biology Organization protein complexed with an RNA hairpin. Nature 372: 432–438 and the Human Frontier Science Program postdoctoral fellowships (R.S.). Pavletich NP, Pabo CO (1991) Zinc finger–DNA recognition: crystal structure of a Zif68-DNA complex at 2.1 Å. 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EMBO J 4: 1609–1614 who is an EMBO Young Investigator 38 EMBO reports VOL 6 | NO 1 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION © 2005 Nature Publishing Group http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EMBO Reports Springer Journals

RNA sequence‐ and shape‐dependent recognition by proteins in the ribonucleoprotein particle

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Abstract

review review RNA sequence- and shape-dependent recognition by proteins in the ribonucleoprotein particle Richard Stefl, Lenka Skrisovska & Frédéric H.-T. Allain Swiss Federal Institute of Technology Zürich, Zürich, Switzerland At all stages of its life (from transcription to translation), an RNA recognize primarily the shape of the RNA or both the sequence and transcript interacts with many different RNA-binding proteins. The the shape. Other types of RNA-binding domains, such as the composition of this supramolecular assembly, known as a ribo- K-homology (KH) domain or the oligonucleotide/oligosaccharide- nucleoprotein particle, is diverse and highly dynamic. RNA-binding binding (OB) fold, have recently been reviewed and are not proteins control the generation, maturation and lifespan of the RNA discussed here (Messias & Sattler, 2004). transcript and thus regulate and influence the cellular function of the encoded gene. Here, we review our current understanding of RNA shape-dependent recognition by double-stranded RBM protein–RNA recognition mediated by the two most abundant RNA- The dsRBM is a 70–75 amino-acid domain with a conserved αβββα binding domains (the RNA-recognition motif and the double-stranded protein topology in which the two α-helices are packed along one RNA-binding motif) plus the zinc-finger motif, the most abundant face of a three-stranded anti-parallel β-sheet (Fig 1; Fierro-Monti & nucleic-acid-binding domain. In addition, we discuss how not only Mathews, 2000; St Johnston et al, 1992). These domains occur mostly the sequence but also the shape of the RNA are recognized by these in multiple copies (up to five) and have so far been found in 388 three classes of RNA-binding protein. eukaryotic proteins, 72 of which are human (data taken from the Keywords: double-stranded RNA-binding motif; RNA-binding SMART database; Letunic et al, 2004). These proteins have an essen- proteins; RNA recognition; RNA-recognition motif; zinc-finger motif tial role in RNA interference, RNA processing, RNA localization, EMBO reports (2005) 6, 33–38. doi:10.1038/sj.embor.7400325 RNA editing and translational repression (Doyle & Jantsch, 2002; Saunders & Barber, 2003). Introduction So far, only three structures of dsRBMs in complex with dsRNA The association of RNA-binding proteins (RBPs) with RNA tran- have been determined (Table 1): a 1.9 Å crystal structure of the sec- scripts begins during transcription. Some of these early-binding ond dsRBM of Xenopus laevis RNA-binding protein A (Xlrbpa2) RBPs remain bound to the RNA until it is degraded, whereas others bound to two coaxially stacked dsRNA molecules, each 10 bp long recognize and transiently bind to RNA at later stages for specific (Ryter & Schultz, 1998); a nuclear magnetic resonance (NMR) struc- processes such as splicing, processing, transport and localization ture of the third dsRBM from the Drosophila Staufen protein in com- (Dreyfuss et al, 2002). The RBPs cover the RNA transcripts and con- plex with a symmetrical GC-rich 12-bp duplex capped by a UUCG trol their fate. Some RBPs function as RNA chaperones (Lorsch, tetraloop (Ramos et al, 2000); and an NMR structure of the dsRBM of 2002) by helping the RNA, which is initially single-stranded, to form Rnt1p (an RNase III homologue from budding yeast) bound to a various secondary or tertiary structures. When folded, these struc- 14-bp RNA duplex capped by an AGAA tetraloop (Wu et al, 2004). tured RNAs, together with specific RNA sequences, act as a signal All three structures have several common features that reveal how a for other RBPs that mediate gene regulation. Here, we review our dsRBM is able to bind to any dsRNA but not to dsDNA, regardless of current structural understanding of protein–RNA recognition medi- its base composition. The dsRBMs interact along one face of the ated by the two most abundant RNA-binding domains, the RNA- RNA duplex through both α-helices and their β1–β2 loop (Fig 1). The recognition motif (RRM) and the double-stranded RNA-binding contacts with the RNA cover 15 bp that span two consecutive minor motif (dsRBM), and by the most abundant nucleic-acid-binding grooves separated by a major groove. In all three structures, the con- motif, the CCHH-type zinc-finger domain. We discuss how these tacts to the sugar-phosphate backbone of the major groove and of three small domains recognize RNA: some bind single-stranded one minor groove (Fig 1) are mediated by the β1–β2 loop and the RNA by direct readout of the primary sequence, whereas others amino-terminal part of α-helix 2. These interactions are non- sequence-specific as they involve 2’-hydroxyls and phosphate oxy- gens and are perfectly adapted to the shape of an RNA double helix. Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology Zürich, ETH-Hönggerberg, CH-8093 Zürich, Switzerland By contrast, the interactions mediated by α-helix 1 are different in all Corresponding author. Tel: +41 (0)1 63 33940; Fax: +41 (0)1 63 31294; three complexes. In the dsRBM of Xlrbpa2, α-helix 1 interacts non- E-mail: [email protected] specifically with the other minor groove of the RNA (Fig 1A), with Submitted 22 September 2004; accepted 26 November 2004 a few contacts to the bases. In the dsRBM of Staufen, α-helix 1 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 1 | 2005 33 © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review target but not its sequence. dsRBMs are highly conserved and have α-helix 2 the same structural framework, but are chemically distinct through variations in key residues. The structure of the dsRBM of Rnt1p in complex with RNA highlights the essential role of the α-helix 1 in α-helix 1 the recognition of structured elements that deviate from regular dsRNA. The α-helix 1 is the least-conserved secondary structure ele- β1–β2 loop ment among various dsRBMs and seems to have a different spatial arrangement relative to the rest of the domain in different dsRBMs. This variability may be an important factor as many biochemical experiments have shown that dsRBM-containing proteins have bind- ing specificity for a variety of RNA structures, such as stem–loops, internal loops, bulges or helices with mismatches (Doyle & Jantsch, 2002; Fierro-Monti & Mathews, 2000; Ohman et al, 2000; Stephens et al, 2004). Clearly, further structures are needed to decipher the extent of RNA shape-dependent recognition by dsRBMs. β-helix 3 RNA sequence- and shape-dependent recognition by an RRM The RRM is the most common RNA-binding motif. It is a small pro- α-helix 2 tein domain of 75–85 amino acids with a typical βαββαβ topology that forms a four-stranded β-sheet packed against two α-helices β1–β2 loop (Mattaj, 1993). RRMs are found in about 0.5%–1% of human genes α-helix 1 (Venter et al, 2001) and are often present in multiple copies (up to six per protein). RRM-domain-containing proteins are involved in many cellular functions, particularly messenger RNA and ribosomal RNA processing, splicing and translation regulation, RNA export and RNA stability (Dreyfuss et al, 2002). So far, ten structures of an RRM in complex with RNA have been determined using either NMR spectroscopy or X-ray crystallography (Table 1). These structures reveal the complexity of protein–RNA recognition mediated by the RRM, which often involves not only protein–RNA interactions but also RNA–RNA and protein–protein Fig 1 | Double-stranded RNA recognition by double-stranded RNA-binding interactions. All ten structures reveal some common features. The motifs. (A) The double-stranded RNA-binding motif (dsRBM) of Xlrbpa2 main protein surface of the RRM involved in the interaction with the bound to dsRNA (Ryter & Schultz, 1998). The α-helix 1 (in red), amino- RNA is the four-stranded β-sheet, which usually contacts two or three terminal part of α-helix 2, and β1–β2 loop recognize non-sequence- nucleotides (exemplified here by the RRM1 of sex-lethal; Fig 2A; specifically the shape of dsRNA. Backbones are coloured blue and light blue for Handa et al, 1999). The nucleotides are located on the surface of the two co-axially stacked duplexes. (B) The dsRBM of Rnt1p bound to the β-sheet, with the bases oriented parallel to the β-sheet plane and stem–loop closed by the AGNN tetraloop (backbone in yellow and bases in often packed against conserved hydrophobic side-chains (usually black; Wu et al, 2004). The α-helix 1, a key element for shape-specific aromatics). These two or three nucleotides are recognized sequence- recognition by dsRBMs, is highlighted in red. The dsRBM of Rnt1p has an specifically by interactions with the protein side-chains of the β-sheet additional carboxy-terminal α-helix 3 (in black), that modulates the and with the main-chain and side-chains of the residues carboxy- conformation of α-helix 1 (Leulliot et al, 2004). Side chains involved in terminal to the β-sheet. Interestingly, it seems that almost all possible intermolecular interactions are shown. sequences (doublets or triplets) can be accommodated on such a surface as the RNA sequences are different in each structure (Table 1). Often, RRM-containing proteins bind more than three interacts with a UUCG tetraloop that caps the RNA double helix. nucleotides and recognize longer single-stranded RNA (for exam- Although the UUCG tetraloop is not a natural substrate of Staufen, ple, poly(A)-binding protein (PAPB; Deo et al, 1999), sex-lethal this finding led to the proposal that α-helix 1 modulates the speci- (Handa et al, 1999), Hu protein D (HuD; Wang & Hall, 2001), ficity of individual dsRBMs (Ramos et al, 2000). Indeed, this was heterogeneous nuclear RNP A1 (hnRNP A1; Ding et al, 1999), recently confirmed by the structure of the dsRBM of Rnt1p bound to nucleolin (Allain et al, 2000; Johansson et al, 2004), RNA its natural RNA substrate (Fig 1B), in which α-helix 1 recognizes the stem–loops U1A (Oubridge et al, 1994), U2B’’ (Price et al, 1998), specific shape of the minor groove created by the conserved AGNN nucleolin (Allain et al, 2000)) or even internal loops (U1A; Allain –9 –1 tetraloop (Wu et al, 2004). The α-helix 1, the conformation of which et al, 1997; Varani et al, 2000), all with high affinity (K ≈ 10 M ). is stabilized by an additional carboxy-terminal α-helix 3 (Fig 1B; In U1A, U2B’’, nucleolin and sex-lethal, two loops between the Leulliot et al, 2004), is tightly inserted into the RNA minor groove secondary-structure elements of the RRM (the β2–β3 loop and and contacts the sugar-phosphate backbone and the two non- the β1–α1 loop) are essential for additional contacts with the RNA conserved tetraloop bases, whereas the conserved A and G bases are (Fig 2B). These loops vary significantly in size and amino-acid not involved in the interactions (Wu et al, 2004). This structure illus- sequence between the different RRMs. In the RRM of CBP20, the trates how this dsRBM recognizes the specific shape of its RNA C- and N-terminal extensions (which are stabilized by the cognate 34 EMBO reports VOL 6 | NO 1 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review Table 1 | Various structures of RNA-binding proteins bound to RNA Complex RNA secondary No. of RBDs in RNA sequence Function Reference; Protein Data Bank structure structures vs in recognized specifically (PDB) ID full-length protein by RRM β-sheet dsRBM type Second dsRBM Duplex 1/3 – hnRNP association, Ryter & Schultz, 1998; 1DI2 of Xlrbpa2 translation repression Third dsRBM Stem–loop 1/5 – mRNA localization, Ramos et al, 2000; 1EKZ of Staufen translation control dsRBM of Rnt1p Stem–loop 1/1 – RNA processing Wu et al, 2004; 1T4L RRM type N-terminal RRM Stem–loop 1/2 CAG Pre-mRNA splicing Price et al, 1998; 1A9N of U2B’’ (in U2B’’– U2A’–RNA complex) N-terminal RRM Stem–loop 1/2 CAC Pre-mRNA splicing Oubridge et al, 1994; 1URN of U1A N-terminal RRM Internal loop 1/2 CAC Pre-mRNA splicing Allain et al, 1997; 1AUD, of U1A Varani et al, 2000; 1DZ5 Two N-terminal Stem–loop 2/4 RRM1-CG Ribosome biogenesis Allain et al, 2000; 1FJE, RRMs of nucleolin RRM2-UC Johansson et al, 2004; 1RKJ Two N-terminal Single strand 2/4 RRM1-AAA Translation initiation Deo et al, 1999; 1CVJ RRM of PABP RRM2-AAA Two RRMs Single strand 2/2 RRM1-UUU Alternative splicing Handa et al, 1999; 1H2T of sex-lethal RRM2-UGU Two N-terminal Single strand 2/3 RRM1-UUU mRNA stability, Wang & Hall, 2001; RRMs of HuD RRM2-UU translation regulation 1FXL, 1G2E RRM of CBP20 Single strand 1/1 m7GpppG Maturation of pre-mRNA Mazza et al, 2002; 1H2V and U-rich snRNA Zinc finger Fourth, fifth and sixth Truncated 3/9 – Transcription regulation Lu et al, 2003; 1UN6 zinc fingers (CCHH- 5S RNA type) of TFIIIA First and second Single strand 2/2 – RNA processing Hudson et al, 2004; 1RGO zinc fingers (CCCH- and degradation type) of TIS11d a b c Two coaxially stacked dsRNA (each 10-bp long). 12-bp duplex capped by a non-physiologically relevant UUCG tetraloop. Consists of loop A, loop E and helices I, IV and V. CBP20, cap-binding protein 20; dsRBM, double-stranded RNA-binding motif; hnRNP, heterogeneous nuclear ribonucleoprotein; HuD, Hu protein D; PABP, poly(A)-binding protein; RBD, RNA-binding domain; Rnt1p, RNase III homologue; RRM, RNA-recognition motif; TFIIIA, transcription factor IIIA; Xlrbpa2, Xenopus laevis RNA-binding protein A. protein CBP80) provide a tight binding pocket for the 5’ capped RNA recognition by zinc fingers RNAs (7-methyl-G(5’)ppp(5’)N, where N is any nucleotide; Fig 2C; CCHH-type zinc-finger domains are the most common DNA-binding Mazza et al, 2002). In proteins that contain several RRMs, high- domain found in eukaryotic genomes. Typically, several fingers are affinity binding can only be achieved by the cooperative binding used in a modular fashion to achieve high sequence-specific recogni- of at least two RRMs to the RNA (for example, in nucleolin tion of DNA (Miller et al, 1985). Each finger displays a ββα protein fold 2+ (Fig 2D), PABP and sex-lethal). In addition to the β-sheet–RNA in which a β-hairpin and an α-helix are pinned together by a Zn ion. contacts, interactions between the inter-domain linker and the DNA-sequence-specific recognition is achieved by the interactions RNA and between the RRMs themselves contribute to the marked between protein side-chains of the α-helix (at position –1, 2, 3 and 6, increase in affinity compared with the binding of the individual for the canonical arrangement) and the DNA bases in the major domain alone. These structures show that the RRM is a platform groove (Fig 3A; Wolfe et al, 2000). However, there is increasing evi- with a large capacity for variation in order to achieve high RNA- dence that zinc fingers are also used to recognize RNA (Finerty & Bass, binding affinity and specificity. For example, it is remarkable that a 1997; Mendez-Vidal et al, 2002; Picard & Wegnez, 1979; Theunissen single domain like nucleolin RRM2 contacts only two nucleotides, et al, 1992). The crystal structure of three zinc fingers (fingers 4–6) of whereas U1A RRM1 contacts 12 nucleotides and the RRM of Y14 transcription factor IIIA (TFIIIA) in complex with a 61-nucleotide frag- (Fribourg et al, 2003) does not contact RNA but rather another pro- ment of the 5S RNA (Lu et al, 2003) provided the first insight into RNA tein. This fascinating plasticity of the RRM explains why it is so recognition by CCHH-type zinc fingers. In this structure, finger 4 binds abundant and why it is involved in so many different biological to loop E, finger 5 to helix V, and finger 6 to loop A (Fig 3B). Finger 4 functions; however, this plasticity makes it difficult to predict how recognizes loop E by specifically interacting with a bulged guanosine the RRM achieves RNA recognition. (Fig 3C) and, similarly, finger 6 recognizes loop A by specifically ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 1 | 2005 35 © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review A C N-terminal C-terminal N-terminal 3' m7GpppG 5' C-terminal B D C-terminal C-terminal Interdomain linker N-terminal β2–β3 loop N-terminal β1–α1 loop Fig 2 | RNA recognition by RNA-recognition motifs. The similarities and differences are highlighted in red and yellow, respectively. (A) RNA-recognition motif 1 (RRM1) of sex-lethal (shown as a ribbon model) interacts with the triplet UUU (shown as a stick model; Handa et al, 1999). The four-stranded β-sheet (in red) recognizes three nucleotides—this is the canonical mode of RRM–RNA interaction. (B) RRM1 of U1A bound to an RNA internal loop (Allain et al, 1997). The four-stranded β-sheet (in red) recognizes three nucleotides. The β2–β3 loop and the β1–α1 loop (in yellow) contact additional RNA residues. (C) RRM of CBP20 complexed with m GpppG (7-methyl-G(5’)ppp(5’)G; Mazza et al, 2002). C- and N-terminal extensions (in yellow) provide additional protein–RNA contacts that creates a specific binding pocket for 5’ capped RNAs. (D) RRMs 1 and 2 of nucleolin bound to an RNA stem–loop (Allain et al, 2000). The four-stranded β-sheet of RRM2 (in red) recognizes only two nucleotides. The interdomain linker (in yellow) participates in the recognition of the hairpin architecture. interacting with two bases (an adenine and a cytosine) that also bulge multiple contacts between basic amino acids of the α-helix and the out from the rest of the RNA (Fig 3B). The specific recognition of the RNA sugar-phosphate backbone (Fig 3D). RNA by both fingers 4 and 6 is achieved by side-chain contacts from In contrast to the above-mentioned CCHH zinc fingers, another the N-terminal parts of the α-helix (at position –1, 1 and 2; Fig 3C). The class of zinc fingers (CCCH-type) was recently found to adopt a dif- interaction of finger 5 with helix V differs from the ones made by fingers ferent fold and to recognize sequence-specifically single-stranded 4 and 6. In this case, finger 5 recognizes a short RNA double helix by RNA (Hudson et al, 2004). In this NMR structure, sequence-specific 36 EMBO reports VOL 6 | NO 1 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION © 2005 Nature Publishing Group Loop E Helix V RNA recognition by RBPs R. Stefl et al. review F6 AB C F5 Loop A Helix V F4 F4 Loop E F5 Fig 3 | DNA vs RNA recognition by CCHH-type zinc fingers. (A) Zinc finger 2 of Zif 268 bound to double-stranded DNA (Pavletich & Pabo, 1991). The α-helix of the zinc finger (in red) inserts into the DNA major groove; base contacts are made from positions –1, 2, 3 and 6 of the α-helix (the protein side-chains are shown as an element-type coloured stick model). The DNA bases that are recognized by the finger are coloured yellow. (B) Overall view of the complex of transcription factor IIIA (TFIIIA) fingers 4–6 (F4–F6) and 61-nucleotide 5S RNA (Lu et al, 2003). The protein and RNA are represented as ribbon models. The bulged bases involved in the recognitions are highlighted in yellow. Cyan balls represent zinc ions. (C) TFIIIA finger 4 (F4) bound to loop E (Lu et al, 2003). The α-helix (in red) of the finger 4 specifically interacts with a guanosine base that bulges out (in yellow); the base contacts are made from the side-chain at position –1, 1 and 2 of the α-helix. (D) TFIIIA finger 5 (F5) bound to helix V (Lu et al, 2003). The α-helix (in red) of finger 5 recognizes the dsRNA shape by non-sequence-specific contacts to the RNA sugar-phosphate backbone. RNA recognition is achieved by a network of intermolecular hydro- regulation. The enormous diversity of interactions observed in pro- gen bonds between the protein main-chain functional groups and tein–RNA complexes indicates that a simple recognition code is the Watson–Crick edges of the bases (Hudson et al, 2004). These unlikely to exist in the world of protein–RNA interactions. However, structures reveal that zinc fingers bind to RNA differently to the way two unifying themes may be inferred from the known complexes: the they do to DNA. The CCHH-type zinc fingers have two modes of recognition of the primary RNA sequence and/or the recognition of RNA binding. First, the zinc fingers interact non-specifically with the the RNA shape by individual RBPs. In a simplistic view, the RRMs, backbone of a double helix, and second, the zinc fingers specifically dsRBMs and CCHH-type zinc fingers seem to be shaped to recognize recognize individual bases that bulge out of a structurally rigid ele- single-stranded RNA, double-stranded RNA and RNA bulges, respec- ment. The CCCH-type zinc fingers show a third mode of RNA binding, tively. However, we have shown here by reviewing several recent in which the single-stranded RNA is recognized in a sequence-spe- protein–RNA complex structures that, the RRM and, to a lesser cific manner. Taken together, zinc fingers represent a unique class of extent, the dsRBMs and the CCHH-type zinc fingers have evolved to nucleic-acid-binding proteins that are capable of a direct readout recognize specifically a rich repertoire of RNAs in terms of length, of the DNA sequence within a DNA double helix, a direct readout of sequence and structure. This is achieved in three ways: first, by the the RNA sequence within single-stranded RNA, and an indirect subtle amino-acid change in variable regions of the domains, namely readout of the RNA as they recognize the shape of the RNA rather the β2–β3 and the β1–α1 loops in the RRM, α-helix 1 in the dsRBM than its sequence. Of course, more structures of CCHH-type and and the α-helix in the zinc fingers; second, by multiplication of the CCCH-type zinc fingers in complex with RNA will need to be domains to achieve higher affinity through cooperative binding; and determined to generalize their mode of RNA recognition. third, by extension of the protein domain. Although more structures still need to be determined, it might soon be possible to predict which Conclusions RBP binds to which RNA, and how it recognizes its target. As a conse- Proteins that contain RNA-binding domains and their interactions quence, post-transcriptional gene expression and its regulation could with RNA have important roles in all aspects of gene expression and be understood and controlled at the atomic level. ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 1 | 2005 37 © 2005 Nature Publishing Group RNA recognition by RBPs R. Stefl et al. review Ohman M, Kallman AM, Bass BL (2000) In vitro analysis of the binding of ACKNOWLEDGEMENTS ADAR2 to the pre-mRNA encoding the GluR-B R/G site. RNA 6: We apologize to authors whose work could not be cited due to space 687–697 constraints. The authors are supported by the Swiss National Science Oubridge C, Ito N, Evans PR, Teo CH, Nagai K (1994) Crystal structure at Foundation (No. 31-67098.01), the Roche Research Fund for Biology at the 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal ETH Zurich (F.H.-T.A.), and the European Molecular Biology Organization protein complexed with an RNA hairpin. Nature 372: 432–438 and the Human Frontier Science Program postdoctoral fellowships (R.S.). Pavletich NP, Pabo CO (1991) Zinc finger–DNA recognition: crystal structure of a Zif68-DNA complex at 2.1 Å. 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EMBO J 4: 1609–1614 who is an EMBO Young Investigator 38 EMBO reports VOL 6 | NO 1 | 2005 ©2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION © 2005 Nature Publishing Group

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EMBO Reports – Springer Journals

Published: Jan 1, 2005

Keywords: double‐stranded RNA‐binding motif; RNA‐binding proteins; RNA recognition; RNA‐recognition motif; zinc‐finger motif

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