title: Sequencing of lariat termini in S. cerevisiae reveals 5' splice sites, branch points, and novel splicing events.
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.
authors.affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Daoming
authors.initials: D
authors.lastname: Qin
authors.affiliation: Center for Research Informatics, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Alissa
authors.initials: A
authors.lastname: Wlodaver
authors.affiliation: Center for Research Informatics, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Andrade
authors.affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Staley
mesh_terms: D001483:Base Sequence; D015966:Gene Expression Regulation, Fungal; D016681:Genome, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D012322:RNA Precursors; D022821:RNA Splice Sites; D012331:RNA, Fungal; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme
keywords: RNA-seq; branch-point; lariat intron; pre-mRNA splicing; splice site