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REVIEW

U2AF homology motifs: protein recognition in the RRM world

¹ Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205, USA; ² Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

	Abstract

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
Recent structures of the heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) have revealed two unexpected examples of RNA recognition motif (RRM)-like domains with specialized features for protein recognition. These unusual RRMs, called U2AF homology motifs (UHMs), represent a novel class of protein recognition motifs. Defining a set of rules to distinguish traditional RRMs from UHMs is key to identifying novel UHM family members. Here we review the critical sequence features necessary to mediate protein–UHM interactions, and perform comprehensive database searches to identify new members of the UHM family. The resulting implications for the functional and evolutionary relationships among candidate UHM family members are discussed.

[Keywords: U2AF; RNA recognition motif; protein–protein interaction; RNA-binding domain; PUMP; splicing factor]

	Structural features of RNA recognition by canonical RRMs

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
The RNA-binding function of the canonical RRM domain has been extensively investigated over the last two decades. The most conserved RRM signature sequence is an eight-residue motif called ribonucleoprotein 1 (RNP1; Adam et al. 1986; Sachs et al. 1986), which has the consensus [RK]-G-[FY]-[GA]-[FY]-[ILV]-X-[FY] (where X is any amino acid). A second six-residue region of homology, called RNP2, is typically located 30 residues N-terminal to RNP1 (Lahiri and Thomas 1986; Dreyfuss et al. 1988), and has the consensus [ILV]-[FY]-[ILV]-X-N-L. Additional conserved amino acids define an 80-residue domain that encompasses the RNA-binding function (Query et al. 1989; Scherly et al. 1989; Birney et al. 1993).

The three-dimensional structure of the canonical RRM domain was first determined for the RRM of U1A (Nagai et al. 1990; Hoffman et al. 1991). The RRM fold is composed of two -helices packed against four antiparallel -strands with topology , which form an / sandwich (Fig. 1). The RNP consensus motifs form two central -strands, with RNP1 in 3 and RNP2 in 1. Because of the alternating side-chain conformations of the pleated -sheet, some of the consensus residues maintain the core fold, whereas others are displayed on the surface for nucleic acid recognition. Structures of single RRMs complexed with RNA have been determined for the U1A-RRM bound to a hairpin loop of U1 snRNA (Oubridge et al. 1994; Price et al. 1998; Deo et al. 1999; Handa et al. 1999; Allain et al. 2000; Wang and Tanaka Hall 2001), and for a ternary complex of the U2 snRNP proteins U2B''-RRM/U2A' with a U2 snRNA hairpin loop (Price et al. 1998). In contrast to the isolated RRM of U1A, in most cases multiple RRMs are observed within a single polypeptide, with an average of two RRMs per protein (Letunic et al. 2004). The structures of several proteins composed of two tandem RRMs complexed with single-stranded RNA oligonucleotides have been determined, including the alternative splicing factor Sxl (Handa et al. 1999), PAB (Deo et al. 1999), pre-rRNA packaging protein nucleolin (Allain et al. 2000), and translation regulatory protein HuD (Wang and Tanaka Hall 2001). A comparison of these six different structures has revealed some common themes, as well as differences, in the mode of canonical RRM/RNA recognition. When all the structures are superimposed, structural equivalent hydrogen-bonds or stacking interactions are observed between single-stranded RNA and residues in the RNP1 and RNP2 motifs. A variety of sequences and RNA conformations are recognized by a variety of complementary hydrogen bonds with specific bases and differing arrangements of single or multiple RRMs.

View larger version (63K): [in this window] [in a new window]   Figure 1. Representative canonical RRM fold, from the structure of the U1A/RNA complex (PDB code 1URN [PDB] ). The N- and C-terminal ends of the RRM are indicated. The position and orientation of the RNA are represented with a ribbon diagram.

	U2AF, the UHM prototype

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

To perform its role in RNA splicing, two central canonical RRM domains of U2AF⁶⁵ recognize the polypyrimidine tract (Py-tract) in the pre-mRNA (Fig. 2). Binding of U2AF⁶⁵ to the Py-tract is strengthened by cooperative protein–protein interactions with SF1 at the upstream BPS (Berglund et al. 1998; Rain et al. 1998) and with U2AF³⁵, which contacts the downstream 3'SS consensus (Merendino et al. 1999; Wu et al. 1999; Zorio and Blumenthal 1999a). The C-terminal UHM domain of U2AF⁶⁵ interacts with the N-terminal domain of SF1 (U2AF⁶⁵-UHM/SF1-ligand; Rain et al. 1998). At the opposite end of the large subunit, the N-terminal domain of U2AF⁶⁵ provides a ligand that interacts with the central UHM domain of U2AF³⁵ (U2AF³⁵-UHM/U2AF⁶⁵-ligand; Zhang et al. 1992; Rudner et al. 1998b). Subsequently, entry of the U2 snRNP displaces SF1 by interacting with the BPS via the U2 snRNA (Nelson and Green 1989; Wu and Manley 1989; Zhuang and Weiner 1989; Query et al. 1994), and with the U2AF⁶⁵ C-terminal domain via the SF3b subunit, SAP155 (Gozani et al. 1998; Habara et al. 1998). Once the U2 snRNP has contacted the pre-mRNA, U2AF is dissociated by conformational rear-rangements of the spliceosome components (Bennett et al. 1992; Chiara et al. 1997). In summary, key protein–protein interactions are mediated by the U2AF⁶⁵-UHM, which interacts with SF1 and subsequently SAP155, and by the U2AF³⁵-UHM, which interacts with the U2AF⁶⁵ N terminus.

View larger version (52K): [in this window] [in a new window]   Figure 2. Diagram of protein–protein interactions mediated by the U2AF heterodimer during the initial stages of pre-mRNA splicing. The U2AF heterodimer (mediated by the U2AF35-UHM/U2AF65-ligand interaction) binding to the poly-pyrimidine tract (Py-tract) and 3'-splice site (3'SS) is facilitated by cooperative interactions between the U2AF65-UHM and SF1 at the branchpoint sequence (BPS). Subsequently, SF1 interactions with the U2AF65-UHM are replaced by SAP155. The N- and C-terminal ends of the proteins are indicated; the conserved ligand Trp residue (W) is also shown (discussed further in the text).

	Structural features of protein–protein interactions by UHMs

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

Three-dimensional structural information revealed unanticipated sequence features of U2AF³⁵-UHM and U2AF⁶⁵-UHM domains that enable interaction with short protein ligands. Despite low primary sequence identity (23%), ligand recognition by the different UHM domains is very similar (Fig. 3). In both the U2AF³⁵-UHM/U2AF⁶⁵-ligand and U2AF⁶⁵-UHM/SF1-ligand structures, a critical Trp residue in the ligand sequence inserts into a tight hydrophobic pocket between the -helices and the RNP1- and RNP2-like motifs (Kielkopf et al. 2001; Selenko et al. 2003). In addition to aliphatic residues, a conserved Arg–X–Phe motif (where X is any amino acid; see below) on the loop connecting the last -helix (Helix B) and -strand of the UHM fold contributes to the Trp-binding pocket. The Arg residue in the loop (U2AF³⁵-Arg 133 or U2AF⁶⁵-Arg 452) forms an intramolecular salt bridge with the last Glu residue of Helix A (U2AF³⁵-Glu 88 or U2AF⁶⁵-Glu 405) that shields one face of the ligand-Trp, whereas the Phe residue (U2AF³⁵-Phe 135 or U2AF⁶⁵-Phe 454) encloses the opposite Trp face. In addition to the extensive interface with the ligand-Trp, a series of acidic residues in Helix A of the UHM interacts with basic residues at the N terminus of the protein ligand. Specifically, electrostatic interactions between U2AF³⁵-Glu 84 and U2AF⁶⁵-Lys 90 as well as U2AF⁶⁵-Asp 401 and SF1-Arg 21 are observed at similar positions for both structures. The essential nature of acidic residues within Helix A, Phe 454, and the Trp-binding pocket was confirmed for the U2AF⁶⁵-UHM/SF1-ligand complex by site-directed mutagenesis of the U2AF⁶⁵-UHM or SF1-ligand followed by pulldown assays (Selenko et al. 2003). Likewise, the U2AF⁶⁵-ligand-Trp 92 was found to contribute two orders of magnitude to the affinity of the U2AF³⁵-UHM/U2AF⁶⁵-ligand complex by isothermal titration calorimetry.

View larger version (46K): [in this window] [in a new window]   Figure 3. Structures of U2AF-UHM/ligand complexes. The UHM is shown in blue, the protein ligand is shown in yellow. Key interacting side chains are drawn in ball-and-stick representation. (A) The U2AF35-UHM bound to the U2AF65-ligand. (B) The U2AF65-UHM bound to the SF1-ligand. (C) Schematic representation of signature UHM protein–protein interactions shared by the two complexes.

The structures of the U2AF³⁵-UHM/U2AF⁶⁵-ligand and U2AF⁶⁵-UHM/SF1-ligand complexes revealed several sequence features that distinguish UHMs from canonical RRM domains. One striking feature of UHM domains is their atypical RNP-like motifs. The first residue of the RNP1-like motif and the second residue of the RNP2-like motif are unusual in that they are exposed on the -sheet surface rather than directly involved in RNA binding. Residues in these positions consist of aliphatic amino acids (U2AF³⁵-Ala 47, Val 110, or U2AF⁶⁵-Cys 379, Cys 429) as opposed to the basic and aromatic residues used for RNA recognition by canonical RRM domains. Other prominent distinguishing sequences include the Arg–X–Phe motif and acidic residues in Helix A (especially U2AF³⁵-Glu 84/Glu 88 and U2AF⁶⁵-Asp 401/Glu 405). As a consequence of the acidic nature of Helix A and lack of a basic RNP1 residue that usually contacts the RNA, the isoelectric points of UHM domains are remarkably low (pI 4.1 for the U2AF³⁵-UHM and pI 4.3 for the U2AF⁶⁵-UHM) compared with the typically basic character of canonical RRMs (pI >9) that function to bind anionic RNA ligands. The majority of the aliphatic residues lining the Trp-binding pocket (including U2AF³⁵-UHM Leu 48, Val 85, Leu 130, and Ile 140 and their U2AF⁶⁵-UHM counterparts Leu 380, Val 402, Leu 449, and Val 459), however, cannot be used to distinguish UHM from canonical RRM domains, because they also serve to preserve the RRM fold (Birney et al. 1993). One exception is the last aliphatic residue of the RNP2 motif (U2AF³⁵-Ile 51 or U2AF⁶⁵-Met 383), which contributes to the Trp-binding pocket and consequently differs from the conserved RNP2-Leu residue within the hydrophobic core of canonical RRM domains. Thus, at least three major sequence differences required for UHM–protein interactions distinguish UHMs from canonical RRM domains: (1) atypical RNP-like motifs, (2) an Arg–X–Phe motif in the last loop, and (3) an acidic character of Helix A.

	Identifying novel UHM family members

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
The discovery of two examples of RRM-like domains with specialized sequence characteristics for protein–protein interactions raised the question of whether the U2AF³⁵-UHM and U2AF⁶⁵-UHM domains represented a larger family of modular protein interaction domains. Examples of proteins with domains similar to the U2AF⁶⁵ or U2AF³⁵-UHM had been previously noted (Kielkopf et al. 2001; Selenko et al. 2003), including the C-terminal homodimerization domain of PUF60, which has previously been referred to as the PUF60, U2AF⁶⁵, MUD2 protein–protein interaction (PUMP) domain (Page-McCaw et al. 1999). Several search strategies were used to further extend the UHM family. An initial consensus pattern for the U2AF⁶⁵, U2AF³⁵, PUF60, and Tat-SF1 UHM domains, defined automatically using the program PRATT (Brazma et al. 1996), proved too stringent as it only matched homologs of these proteins in a Scan-ProSite search of the SWISS-PROT/TrEMBL databases (Gattiker et al. 2002). Therefore, a target pattern ([ILM-VFC]-X-[LIFV]-X-[NSHT]-[ILMVC]-X(6,40)-[VLIT]-X(2)-[ED]-X(4,5)-G-X-[IVA]-X(4)-[VIL]-X(4,25)-[GV]-X-[VIAL]-[FY]-[VIL]-X-[FYC]-X(6,12)-[AC]-[LVMIC]-X-X-[LMIF]-X-[NG]-R-[WYKM]-[FY]-X-G-X(4,8)-[IVL]) was defined manually based upon conserved residues that either maintain the RRM-like fold (Birney et al. 1993) or mediate protein–protein interactions in the structures of U2AF³⁵-UHM/U2AF⁶⁵-ligand and U2AF⁶⁵-UHM/SF1-ligand (Kielkopf et al. 2001; Selenko et al. 2003). A search of the SWISS-PROT/TrEMBL databases with this target pattern identified several novel UHM candidates. The UHM family was further extended by manually inspecting RRM family alignments (Prosite PS50102) and the results of iterative PHI-PSI BLAST searches (Altschul et al. 1997) for similarities to the signature Arg–X–Phe motif observed in the last loop of the prototype U2AF-UHM domains.

Sequence comparisons revealed that the principal features that distinguish UHM candidates from canonical RRMs are conserved among 12 novel UHM candidates (Table 1; Fig. 4A), including (1) poor conservation of amino acids in the RNP1- and RNP2-like consensus motifs that would normally bind RNA (first/third and second positions, respectively); (2) an Arg–X–Phe motif in the last loop of the RRM-like fold; and (3) conserved acidic residues in the predicted Helix A and a low isoelectric point (average pI 4.5). Seven additional UHM candidates displayed a subset of the UHM characteristics. To further investigate the evolutionary relationship among members of the UHM and RRM families, a phylogenetic tree of the candidates was constructed using neighbor joining with correction for multiple substitutions (Fig. 4B; Thompson et al. 1997). A comparison with canonical RRMs whose role in RNA recognition has been established by structure determination (including U1A, SXL, PAB, HuD, and nucleolin) revealed that the 12 convincing UHM candidates occupy a phylogenetic branch distinct from canonical RRMs that diverged from a common ancestral domain. The dendrogram also confirms that several of the putative UHM candidates (i.e., those that displayed only a subset of the UHM characteristics) are more closely related to canonical RRM domains than to U2AF or other UHM candidates, indicating that these proteins may have independently evolved UHM-like sequence motifs. Additional proteins from diverse eukaryotes displayed UHM signature sequences, but were considered homologs of other UHM candidates based on high sequence identity (Table 2). Given the difficulty of distinguishing UHMs from canonical RRM domains based on primary sequence comparisons alone, additional UHM protein interaction domains may be hidden within the RRM superfamily.

View larger version (120K): [in this window] [in a new window]   Figure 4. Identification of candidate UHM-containing proteins. (A) Structure-based alignment of candidate UHM sequences, U2AF35-UHM, U2AF65-UHM, and representative canonical RRMs whose structures in complex with RNA are known. Structures were aligned using the program TOPP from the CCP4 suite (Collaborative Computational Project, no. 4, 1994). Secondary structure elements are indicated above the sequences. The positions of key residues that distinguish UHM from RRM domains are highlighted yellow. Consensus UHM residues including acidic residues and the Arg–X–Phe loop are red. Residues in the RNP-like motifs that match the canonical RRM consensus are blue. (B) Phylogenetic tree of candidate UHM domains.

The 12 candidate UHM domains are found in the context of a variety of domain arrangements within their protein sequences; a subset is detailed in Table 3. With the exception of the central URP-UHM, the UHM domains often occur near the C terminus of the candidate proteins, providing an exposed position to facilitate molecular recognition. Many of the UHM candidates also contain motifs frequently observed in splicing factors, such as canonical RRMs, arginine–serine (RS) domains, zinc fingers, and Gly-rich regions. Additional unexpected domains are also observed, including the LAP2-Emerin-Man1 (LEM) protein–protein interaction domain of MAN1 and kinase domain of KIS.

	Specificity of protein–protein interaction by UHM domains

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
As stated above, the structures of the U2AF³⁵-UHM and U2AF⁶⁵-UHM complexed with their ligands revealed remarkably similar modes of protein interaction. Notably, both the U2AF⁶⁵ and SF1 ligands bind their respective UHM domains via a similar arrangement of basic and Trp residues. A search for the ligand consensus pattern [RK]-X-[RK]-W, shared by both the SF1 and U2AF⁶⁵ ligands, found >1000 matches within the SWISS-PROT database, indicating that predicting protein ligands of candidate UHMs is impractical without further experimental information to identify their functional binding partners. Given that all 12 compelling UHM candidates possess the signature sequences predicted to recognize ligands containing the [RK]-X-[RK]-W motif, it remains an open question whether each UHM domain specifically recognizes a single target, or could promiscuously interact with the intended ligand of a different UHM in the absence of temporal or spatial regulation. Toward answering this question, the U2AF⁶⁵-UHM has been found not to interact with the N terminus of the U2AF⁶⁵-ligand in two-hybrid assays (Tronchere et al. 1997) and pull-down experiments (C. Kielkopf, unpubl.), suggesting that individual UHMs may, indeed, specifically recognize a unique target. By analogy with other modular peptide binding domains (Pawson and Nash 2003), UHM sequences flanking the Trp-binding pocket may ensure specific and directional interactions (N-to-C orientation) with the ligand.

In support of this analogy, structure-based modeling suggests that variation of the central X residue in the UHM Arg–X–Phe loop may provide one mechanism for UHM recognition of diverse C-terminal ligand sequences. Several of the UHM candidates (hURP, PUF60, Tat-SF1, and HCC1) share a Trp residue within the Arg–X–Phe loop that is essential in the U2AF³⁵-UHM for specific recognition of C-terminal U2AF⁶⁵-ligand-Pro residues (Kielkopf et al. 2001). Other UHM sequences vary from similar Arg–Tyr–Phe motifs (SPF45, DRT111, UAP2, CUS2, and PAD1) to divergent Lys (U2AF⁶⁵) and Met (KIS) residues. Besides recognizing the ligand C terminus via the Arg–X–Phe loop, distinct U2AF⁶⁵-UHM or U2AF³⁵-UHM residues make specific contacts with N-terminal ligand residues. In particular, the bulky U2AF⁶⁵-ligand Tyr 91 stacks against unique U2AF³⁵ aromatic residues (Tyr 52 and Phe 81), and forms a specific hydrogen-bond with His 77 of the U2AF³⁵-UHM (Kielkopf et al. 2001). Similar or identical residues in the URP-UHM (Phe 206, Phe 239, and Gln 235) suggest that a bulky, hydrophobic residue preceding the consensus ligand-Trp would be recognized in an analogous manner, consistent with an interaction between hURP and U2AF⁶⁵ in pull-down and yeast two-hybrid assays (Tronchere et al. 1997). The smaller size of the corresponding U2AF⁶⁵-UHM residues (Ile 398 and Val 384) would leave the hydrophobic side chain of a ligand-Tyr in an unfavorable, solvent-exposed environment (Selenko et al. 2003). Considering the variety of cellular roles played by UHM candidates and the consequent requirement to recognize diverse protein ligands, it will be important to determine whether variation in the positions corresponding to U2AF³⁵ Tyr 52, Phe 81, and His 77, and the central position of the Arg–X–Phe loop enables recognition of distinct ligand sequences by UHM domains.

In addition to recognizing short peptide ligands, UHM domains can self-associate to form protein homodimers. For example, the PUF60-UHM domain interacts with itself in two-hybrid assays (Poleev et al. 2000) and forms SDS-resistant homodimers during electrophoresis (Page-McCaw et al. 1999). The U2AF³⁵-UHM has been shown to form weak homodimers by gel filtration, analytical ultracentrifugation, dynamic light scattering (Kielkopf et al. 2001), and two-hybrid assays (Wentz-Hunter and Potashkin 1996), whereas homodimers of the U2AF⁶⁵-UHM have not been observed (Tronchere et al. 1997). Homo- or heterotypic oligomerizations also have been observed for classical protein–protein interaction domains, with several different effects on ligand recognition. For example, the nNOS-PDZ/syntrophin heterodimer prohibits peptide recognition (Hillier et al. 1999), whereas GRIP or Shank PDZ homodimers leave the peptide-binding pockets free (Im et al. 2003a,b) and the Eps8-SH3 homodimer alters the ligand specificity (Kishan et al. 1997). Although a U2AF³⁵-UHM homodimer can be modeled with the solvent exposed Arg–Trp–Phe loop binding to the Trp-binding site on a second UHM domain, alternative interfaces are possible that would allow the oligomer to simultaneously recognize peptide ligands, as observed for established protein–protein interaction domains.

	Do UHM domains recognize RNA?

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
Modeling of the U2AF-UHM/ligand structures with RNA has revealed that peptide binding to the helical surface of the RRM-like fold is not predicted to physically interfere with putative RNA interactions on the opposite -sheet face (Kielkopf et al. 2001; Selenko et al. 2003). Although the U2AF³⁵-UHM/U2AF⁶⁵-ligand complex binds RNA weakly (Kd >6 µM), accessory protein factors and adjacent domains in the full-length U2AF³⁵ sequence (e.g., flanking zinc fingers and an RS domain) are required to assist the weak interaction (Rudner et al. 1998a; J. Valcarcel, pers. comm.). Likewise, the U2AF⁶⁵-UHM domain is not required for Py-tract recognition (Banerjee et al. 2003), and does not appear to interact with RNA (Selenko et al. 2003). These results indicate that UHM domains are not likely to be involved in RNA interactions.

Instead, the UHM family has evolved sequence characteristics that have no benefit for RNA binding, while optimizing the interaction with peptide ligands. In most canonical RRM-RNA structures, conserved aromatic Phe/Tyr residues at the third RNP1 position or second RNP2 position stack with RNA bases or sugars, and a basic Arg/Lys residue at the first position of the RNP1 motif frequently forms a salt bridge with the phosphate backbone (Fig. 5A; Oubridge et al. 1994; Price et al. 1998; Deo et al. 1999; Handa et al. 1999; Allain et al. 2000; Wang and Tanaka Hall 2001). In contrast, the corresponding U2AF³⁵-UHM (Fig. 5B) and U2AF⁶⁵-UHM (Fig. 5C) residues are replaced with aliphatic substitutions that are not predicted to interact favorably with RNA. Moreover, UHMs display unexpectedly low isoelectric points for optimal binding of basic peptides.

View larger version (61K): [in this window] [in a new window]   Figure 5. RNA recognition by an RRM domain compared with the U2AF-UHM domains. The UHM or RRM domains are shown in blue, protein ligands are yellow, and RNA ligands are purple. (A) The U1A-RRM recognizing an RNA oligonucleotide. The RNP1-Arg recognizes the RNA phosphates, a nucleotide base stacks on RNP2-Tyr, and RNP2-Leu is folded in the hydrophobic core. (B) The U2AF35-UHM/U2AF65-ligand complex (PDB code 1JMT [PDB] ) rotated 180° about the Y-axis with respect to Figure 3 to show the putative RNA interaction surface. (C) Similar view of the U2AF65-UHM/SF1-ligand structure (PDB code 1O0P [PDB] ). The RNP2-Ile packs with the ligand-Trp. The small aliphatic residues in the RNP-like motifs are predicted to be unable to form favorable RNA interactions. Schematic representation of RNP residues that differ between UHMs compared with canonical RRMs is shown to the right.

	Conclusions

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
The canonical RRM domain was a relatively late evolutionary addition to the array of RNA-binding folds that emerged in response to the needs of complex pre-mRNA processing pathways (Anantharaman et al. 2002). As processes that were originally based on the RNA world became progressively more regulated and reliant on protein interactions, the RRM fold further developed specialized sequence characteristics for protein recognition to form the UHM subfamily. These UHM signature sequences included divergent residues in the RNP-like motifs, an Arg–X–Phe loop sequence, and key acidic residues that collectively recognized the Trp residue and positive charge of the protein ligand. Convincing UHM candidates have been discovered in association with a variety of fundamental cellular processes, ranging from pre-mRNA splicing to transcription, DNA repair, and signal transduction. The large number of proteins that share the signature protein–protein interaction residues of UHM domains supports the proposal that the U2AF-UHMs represent a novel family of modular protein interaction domains.

Because protein interaction domains are attractive modules for communication among a network of pathways, the UHM domain may be an evolutionary extension of RRMs that couples pre-mRNA processing with other nuclear processes. Protein recognition by so-called RNA-binding domains is an emerging theme in molecular recognition. An early example of RRM–protein interactions was observed in the structure of U2B''/U2A', in which the -helical surface of the U2B''-RRM interacts with the U2A' leucine-rich repeat motif (Price et al. 1998). Several recent structures of the -sheet surfaces of heterodimeric RRM domains interacting with -helical protein ligands have revealed a second mode of RRM–protein recognition distinct from that of UHM/protein complexes (Fribourg et al. 2003; Lau et al. 2003; Shi and Xu 2003; Kadlec et al. 2004). In addition to distinguishing protein recognition domains within the RRM family, a growing list of fold families such as the Sterile -Motif (SAM; Kim and Bowie 2003), LEM (Cai et al. 2001; Laguri et al. 2001), Pumilio/HEAT-repeat domains (Wang et al. 2002), and zinc fingers (Morgan et al. 1997) have been found to bind either nucleic acids or protein ligands through slight variations of a common scaffold. Furthermore, RS domains have been shown to contact the pre-mRNA during splicing (Valcarcel et al. 1996; Shen et al. 2004), and have also been reported to mediate protein–protein interactions (Wu and Maniatis 1993). Because a major goal of the "postgenomic" era is the ability to predict protein functions even in the absence of corroborating experimental results (Thornton et al. 2000), it will be essential to compile a lexicon of signature sequences, such as those that distinguish UHMs from canonical RRMs, for other fold families whose members play diverse functional roles.

	Acknowledgments

Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
We thank S. Evans for editorial assistance, and J. Bender, M. Matunis, M. Swenson, and J. Wedekind for careful reading of the manuscript. Funding for C.L.K. is provided by the Johns Hopkins University Center for AIDS Research grant #P30 AI42855.

	Footnotes

 
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1206204.

Corresponding authors.

³ E-MAIL ckielkop{at}jhsph.edu; FAX (410) 955-2926.

⁴ E-MAIL michael.green{at}umassmed.edu; FAX (508) 856-5473.

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Top Abstract Structural features of RNA... U2AF, the UHM prototype Structural features of protein... Identifying novel UHM family... Specificity of protein-protein... Do UHM domains recognize... Conclusions Acknowledgments References

 
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