Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo

Laboratory of Molecular Medicine, Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 06/2012; 19(7):719-21. DOI: 10.1038/nsmb.2327
Source: PubMed


We present the first large-scale identification of lariats-the transient branched introns that are released as a byproduct of pre-mRNA splicing. The locations of the branchpoints in these introns provide insight into the early steps of splicing. From this data set, we have developed a comprehensive model of 3' splice-site selection, identified new mechanisms of alternative splicing and mapped the distribution of splicing factors around branchpoints.

    • "We restricted our analysis to the 59,359 high-confidence branchpoints confirmed by mismatch sequencing errors that correspond to ;17.4% of introns occurring within 10,773 genes (24.8% of total). Branchpoint annotations exhibited several previously described features (Gao et al. 2008; Corvelo et al. 2010; Taggart et al. 2012), including a restricted distribution upstream of the 39 splice site, with 90% of branchpoints occurring within 19 to 37 (median 25) nucleotides upstream (Fig. 4B). The majority of branchpoint nucleotides correspond to adenine (78.4%), with lower frequency selection of cytosine (8.4%), uracil (8.4%), and guanine (4.7%) nucleotides that have been shown to function, albeit with lower efficiency, as branchpoints (Fig. 4A; Reed and Maniatis 1988). "
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    ABSTRACT: During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations. © 2015 Mercer et al.; Published by Cold Spring Harbor Laboratory Press.
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    ABSTRACT: After transcription of a eukaryotic pre-mRNA, its introns are removed by the spliceosome, joining exons for translation. The intron products of splicing have long been considered 'junk' and destined only for destruction. But because they are large in size and under weak selection constraints, many introns have been evolutionarily repurposed to serve roles after splicing. Some spliced introns are precursors for further processing of other encoded RNAs such as small nucleolar RNAs, microRNAs, and long noncoding RNAs. Other intron products have long half-lives and can be exported to the cytoplasm, suggesting that they have roles in translation. Some viruses encode introns that accumulate after splicing and play important but mysterious roles in viral latency. Turnover of most lariat-introns is initiated by cleavage of their internal 2'-5' phosphodiester bonds by a unique debranching endonuclease, and the linear products are further degraded by exoribonucleases. However, several introns appear to evade this turnover pathway and the determinants of their stability are largely unknown. Whereas many stable intron products were discovered serendipitously, new experimental and computational tools will enable their direct identification and study. Finally, the origins and mechanisms of mobility of eukaryotic introns are mysterious, and mechanistic studies of the intron life cycle may yield new insights into how they arose and became widespread. Conflict of interest: The author has declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.
    No preview · Article · Jul 2013 · WIREs RNA
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    ABSTRACT: Alternative splicing is a potent regulator of gene expression that vastly increases proteomic diversity in multicellular eukaryotes and is associated with organismal complexity. Although alternative splicing is widespread in vertebrates, little is known about the evolutionary origins of this process, in part because of the absence of phylogenetically conserved events that cross major eukaryotic clades. Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing. Remarkably, an evolutionary analysis of four of the exons identified here as subject to skipping in S. pombe reveals high sequence conservation and perfect length conservation with their homologs in scores of plants, animals, and fungi. Moreover, alternative splicing of two of these exons have been documented in multiple vertebrate organisms, making these the first demonstrations of identical alternative-splicing patterns in species that are separated by over 1 billion y of evolution.
    Preview · Article · Jul 2013 · Proceedings of the National Academy of Sciences
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