U2 auxiliary factor (U2AF) is an essential splicing factor that recognizes the 3' splice site and recruits the U2 snRNP to the branch point. The X-ray structure of the human core U2AF heterodimer, consisting of the U2AF35 central domain and a proline-rich region of U2AF65, has been determined at 2.2 A resolution. The structure reveals a novel protein-protein recognition strategy, in which an atypical RNA recognition motif (RRM) of U2AF35 and the U2AF65 polyproline segment interact via reciprocal "tongue-in-groove" tryptophan residues. Complementary biochemical experiments demonstrate that the core U2AF heterodimer binds RNA, and that the interacting tryptophan side chains are essential for U2AF dimerization. Atypical RRMs in other splicing factors may serve as protein-protein interaction motifs elsewhere during spliceosome assembly.
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.The processes of RNA splicing, transport, capping, editing, and polyadenylation are heavily dependent on protein factors that recognize the pre-mRNA and assemble the appropriate pre-mRNA processing complexes. Surprisingly, the many different protein factors that guide pre-mRNA modification pathways are composed of a limited number of conserved, modular RNA-binding domains (Burd and Dreyfuss 1994). Of these, the RNA recognition motif (RRM) domain is by far the most abundant type of eukaryotic RNA-binding motif. In addition to associations between protein and RNA, protein-protein interactions are essential to recruit catalytic components to sites of RNA modification and to coordinate pre-mRNA processing with other cellular pathways. Interestingly, traditional protein interaction domains, such as SH2, SH3, and WW motifs, are rarely observed in pre-mRNA processing factors (e.g., see Shatkin and Manley 2000;Zhou et al. 2002), implying that the ability to interact with other proteins may reside in the sequences previously thought to be involved in RNA binding. Consistent with this idea, recent structures of the heterodimeric splicing factor U2 snRNP auxiliary factor (U2AF) have revealed two unexpected examples of RRM-like domains with specialized features for protein recognition (Kielkopf et al. 2001;Selenko et al. 2003). In light of this structural information, we call these unusual RRMs U2AF homology motifs (UHMs) to reflect their distinct role in protein recognition. Here, the critical sequence features necessary to mediate protein-UHM interactions are reviewed and formulated in a manner that has permitted a comprehensive database search designed to identify members of the UHM family. The resulting implications for the functional and evolutionary relationships among candidate members of the UHM family are discussed. This review represents a first step toward distinguishing canonical RRMs from UHMs, and thereby contributes toward a major goal of the postgenomic era (Thornton et al. 2000): to convert genomic sequences into testable functional hypotheses. Structural features of RNA recognition by canonical RRMsThe 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-resid...
Small molecules that target specific DNA sequences offer a potentially general approach for the regulation of gene expression. Pyrrole-imidazole polyamides represent the only class of synthetic small molecules that can bind predetermined DNA sequences with affinities and specificities comparable to DNA binding proteins. Antiparallel side-by-side pairings of two aromatic amino acids, imidazole (Im) and pyrrole (Py), distinguish G.C from C.G, and both from A.T/T.A base pairs. A high resolution X-ray crystal structure of a four-ring pyrrole-imidazole polyamide specifically bound as a dimer to a six-base pair predetermined DNA site reveals a structural framework of hydrogen bonds and interactions with the walls of the minor groove that underlies the pairing rules for DNA recognition.
The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.
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