U2AF adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA and Howard Hughes Medical Institute and Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2013; 41(6). DOI: 10.1093/nar/gkt046
Source: PubMed


Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential
pre-mRNA splicing factor U2AF65 is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3′ splice sites while adapting
to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF65 recognizes degenerate Py tracts, we determined six crystal structures of human U2AF65 bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts,
we also determined two baseline structures of U2AF65 bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest
that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal
RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant
U2AF65 for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated
that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF65 conformations. Our results highlight both local and global conformational selection as a means for universal 3′ splice site
recognition by U2AF65.

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    • "The side-by-side cross-validation of the structural and the iCLIP data hints that the two RRMs of U2AF 65 indeed bind in tandem on their cellular target sites, with their relative orientation preserved. Together, the examples of these two RBPs suggest that combining highresolution structural and genomic data allows to describe cellular RBP: Figure 7Comparison between the structurally derived sequence-specificity of hnRNP C RRM (A) and U2AF 65 RRM12 (B) (Jenkins et al., 2013), and their transcriptome-wide CLIP consensus sequences (K€onig et al., 2010;Zarnack et al., 2013). (A) The number of intermolecular hydrogen bonds observed for each RRM pocket is compared to the uridine frequency observed in aligned cross-linked sites. "
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    ABSTRACT: RRM-containing proteins are involved in most of the RNA metabolism steps. Their functions are closely related to their mode of RNA recognition, which has been studied by structural biologists for more than 20 years. In this chapter, we report on high-resolution structures of single and multi RRM-RNA complexes to explain the numerous strategies used by these domains to interact specifically with a large repertoire of RNA sequences. We show that multiple variations of their canonical fold can be used to adapt to different single-stranded sequences with a large range of affinities. Furthermore, we describe the consequences on RNA binding of the different structural arrangements found in tandem RRMs and higher order RNPs. Importantly, these structures also reveal with very high accuracy the RNA motifs bound specifically by RRM-containing proteins, which correspond very often to consensus sequences identified with genome-wide approaches. Finally, we show how structural and cellular biology can benefit from each other and pave a way for understanding, defining, and predicting a code of RNA recognition by the RRMs. © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jun 2015 · Methods in enzymology
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    • "The spliced mRNA species in HIV-1 infected cells are env/vpu, nef, rev, vpr, and vif. The binding of a 35 kDa subunit stabilizes the binding of U2AF65 with the polypyrimidine tract (PPT) of introns during spliceosome assembly [35,44]. However, there are no reports that altered expression of U2AF65 contributes to HIV-1 viral progression or susceptibility of transmission. "
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    ABSTRACT: Pre-mRNA splicing is a critical event in the gene expression pathway of all eukaryotes. The splicing reaction is catalyzed by the spliceosome, a huge protein-RNA complex that contains five snRNAs and hundreds of different protein factors. Understanding the structure of this large molecular machinery is critical for understanding its function. Although the highly dynamic nature of the spliceosome, in both composition and conformation, posed daunting challenges to structural studies, there has been significant recent progress on structural analyses of the splicing machinery, using electron microscopy, crystallography, and NMR. This review discusses key recent findings in the structural analyses of the spliceosome and its components, and how these findings advance our understanding of the function of the splicing machinery.
    No preview · Article · Jun 2013 · Protein Science
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