Endogenous U2·U5·U6 snRNA complexes in <i>S. pombe</i> are intron lariat spliceosomes

Chen, Weijun; Shulha, Hennady P.; Ashar-Patel, Ami; Yan, Junkai; Green, Karin M.; Query, Charles C.; Rhind, Nick; Weng, Zhiping; Moore, Melissa J.

doi:10.1261/rna.040980.113

Cited by 43 publications

(60 citation statements)

References 65 publications

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“…A previous study identified an interaction between S. cerevisiae Drn1 and the Nineteen Complex component Ntc40/Cwc2 (Hazbun et al 2003); paralogs of Drn1 in S. pombe (Cwf19p and Mug161p) copurify with Nineteen Complex components (Ohi et al 2002;Ren et al 2011;Chen et al 2014); and human Drn1 paralogs (Cwf19l1 and Cwf19l2) copurify with spliceosomes (Rappsilber et al 2002).…”

Section: Drn1 Interacts With Spliceosomal Components and Branched Rnamentioning

confidence: 93%

A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Garrey

Katolik

Prekeris

et al. 2014

RNA

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Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudometallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5 ′ -exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.

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Section: Drn1 Interacts With Spliceosomal Components and Branched Rnamentioning

confidence: 93%

A homolog of lariat-debranching enzyme modulates turnover of branched RNA

Garrey

Katolik

Prekeris

et al. 2014

RNA

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“…An alternative to enriching for lariats by stabilization is to enrich for lariats by coimmunoprecipitation with the spliceosome; in extracts from wild-type cells, immunoprecipitation of endogenous spliceosomes coprecipitates lariats (e.g., Small et al 2006;Chen et al 2014). To test whether lariat enrichment by immunoprecipitation of the spliceosome enables the application of LIT-seq to wild-type cells, we immunoprecipitated spliceosomes using antibodies against Prp16, one of the factors that associates with the spliceosome at the lariat intermediate stage and through coimmunoprecipitation enriches for lariat intermediates (Schwer and Guthrie 1991).…”

Section: Lit-seq Can Be Applied To Wild-type Cellsmentioning

confidence: 99%

“…S7). Because endogenous spliceosomes have been immunopreciptated previously from other organisms, such as human cells (e.g., Girard et al 2012), and transcripts bound to endogenous spliceosomes have already been identified (Volanakis et al 2013;Chen et al 2014;Nojima et al 2015), we anticipate that LIT-seq can be applied broadly to other organisms to efficiently, specifically, and directly monitor splicing site choice genome-wide and with single-nucleotide resolution.…”

Section: Future Applications Of Lit-seqmentioning

confidence: 99%

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

Qin

Huang

Wlodaver

et al. 2015

RNA

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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 suboptimalespecially 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.

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“…RNA-RNA interactions are mainly responsible in spliceosome remodeling and catalytic core conformation [11][12][13] and numerous proteins are also required for assembly and splicing catalysis and splicing fidelity as well [14][15][16]. Participating factors of mainly human and yeast have been determined by mass spectrometry (MS) of in vitro assembled and affinity-purified spliceosomal complexes of model pre-mRNAs [17][18][19][20][21][22][23][24][25][26][27][28][29]. Over 150 proteins have been recruited in such complexes and this repertoire has been extended when the analysis was carried out from yeast, human and chicken cellular spliceosomes [30].…”

Section: Introductionmentioning

confidence: 99%