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

Taggart, Allison J; DeSimone, Alec M.; Shih, Janice S; Filloux, Madeleine E.; Fairbrother, William G.

doi:10.1038/nsmb.2327

Cited by 115 publications

(169 citation statements)

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“…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).…”

Section: Branchpoint Featuresmentioning

confidence: 95%

“…In contrast, only a few hundred corresponding branchpoints have been collectively described in literature (Taggart et al 2012;Bitton et al 2014), and the identification and analysis of branchpoints has been absent from ambitions for a full catalog of functional genetic elements.…”

Section: Discussionmentioning

confidence: 99%

“…Further complicating de novo branchpoint predictions are reports that some introns have multiple branchpoints, branchpoints distal from the 39 splice site, or without the canonical adenine base at the branchpoint nucleotide (Gao et al 2008;Taggart et al 2012;Bitton et al 2014). However, during the preparation of cDNA, reverse transcriptase can traverse the 59SS/branchpoint junction, thereby generating a cDNA with the 59 intronic sequence juxtaposed to the sequence immediately upstream of the branchpoint (Vogel et al 1997).…”

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confidence: 99%

“…Mutations that abolish the branchpoint nucleotide can result in exon skipping and aberrant splicing and cause human disease (Padgett 2012;Singh and Cooper 2012). Despite such importance, only a few hundred human branchpoints have been identified, thereby preventing a detailed analysis of this basal splicing element (Gao et al 2008;Taggart et al 2012;Bitton et al 2014).…”

mentioning

confidence: 99%

“…The split and inverted alignment of the resultant cDNA identifies not only the 59 donor splice site but also the branchpoint nucleotide ( Fig. 1; Taggart et al 2012;Bitton et al 2014), yet such events are encountered so rarely as to have hitherto prevented efficient and comprehensive genome scale annotation.…”

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confidence: 99%

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Genome-wide discovery of human splicing branchpoints

et al. 2015

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During the splicing reaction, the 59 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 39 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.[Supplemental material is available for this article.]The majority of human genes are spliced, a process whereby introns are removed from the nascent RNA and the remaining exonic sequence joined together into a mature RNA transcript. In addition, alternative splicing generates complex networks of isoforms from human gene loci and plays a major role in shaping the diversity of the transcriptome (Kapranov et al. 2005;Gerstein et al. 2007;Djebali et al. 2012).Splicing occurs in the spliceosome, a large ribonucleoprotein complex that recognizes at least three genetic elements within each intron: the 59 splice site (59SS), the 39 splice site (39SS), and the branchpoint (Will and L€ uhrmann 2011). RNU2-1, the U2 spliceosomal RNA (snRNA) base pairs to the sequence surrounding the unpaired branchpoint nucleotide, which then undergoes transesterification with the 59 end of the intron to form a closed lariat structure. The spliceosome then scans for the downstream 39 splice site, which undergoes a second trans-esterification reaction to join together the two exon ends and excise the intron lariat (Fig.

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Section: Branchpoint Featuresmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Genome-wide discovery of human splicing branchpoints

et al. 2015

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Messenger RNA Splicing Signals

Cygan

Taggart

Fairbrother

et al. 2017

Encyclopedia of Life Sciences

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Splicing is a post‐transcriptional processing step in which intervening sequences (introns) are excised and coding sequences (exons) are ligated together to create the mature mRNA (messenger ribonucleic acid) molecule. It is a sequential process that is facilitated by the information in the RNA (ribonucleic acid) sequence (splicing regulatory elements/signals) and numerous RNA‐binding proteins (trans factors). Splicing occurs through two biochemical steps, and is catalysed by a large ribonucleoprotein known as the spliceosome. The spliceosomal machinery recognises the core splicing signals and assembles in a stepwise fashion on the premRNA molecule. These signals include the obligate 5′ splice site, 3′ splice site and branch site sequence, as well as enhancer and silencer sequences which functionally interact with RNA binding proteins. A current research application in the field uses computational approaches to study splicing signals and predict the effect of single nucleotide variations on message processing. Key Concepts Splicing is a post‐transcriptional process in which certain sections of RNA (known as introns) are removed and other sections (known as exons) are ligated together, producing the mature mRNA molecule. Splicing is catalysed by a large macromolecular machine known as the spliceosome. The major elements required for splicing include the 5′ splice site (5′ss), the 3′ splice site (3′ss), the polypyrimidine tract and the branch site. Most introns begin with a ‘GT’ sequence and end with an ‘AG’ sequence; however, there exists a class of intron with different boundary sequences that are recognised by a parallel spliceosome. Outside of these major elements, there are additional enhancer and suppressor signals that interact with RNA‐binding proteins (RBPs) and affect splicing outcome. Different combinations and positions of binding of RBPs can result in different splicing outcome. Scientists do not yet have a complete understanding of all of the different splicing signals, and this topic is an area of active research. Researchers are using computational machine learning approaches to predict the effects of sequence variation on splicing outcome. Researchers are also using new methods of sequencing to determine which RNA sequences the RBPs are recognising.

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Lives that introns lead after splicing

Hesselberth

2013

WIREs RNA

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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.

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

Cited by 115 publications

References 21 publications

Genome-wide discovery of human splicing branchpoints

Genome-wide discovery of human splicing branchpoints

Messenger RNA Splicing Signals

Lives that introns lead after splicing

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