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RNA. 2016 Feb;22(2):237-53. doi: 10.1261/rna.052829.115. Epub 2015 Dec 8.

Sequencing of lariat termini in S. cerevisiae reveals 5' splice sites, branch points, and novel splicing events.

Author information

  • 1Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
  • 2Center for Research Informatics, University of Chicago, Chicago, Illinois 60637, USA.

Abstract

Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimal-especially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5' splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3' termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3' to 5' RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

KEYWORDS:

RNA-seq; branch-point; lariat intron; pre-mRNA splicing; splice site

PMID:
26647463
PMCID:
PMC4712674
DOI:
10.1261/rna.052829.115
[PubMed - indexed for MEDLINE]
Free PMC Article
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