Department of Biochemistry and Molecular Pharmacology; UMMS Proteomics and Mass Spectrometry Facility; Program in Bioinformatics and Integrative Biology
Blotting, Northern; Blotting, Western; High-Throughput Nucleotide Sequencing; Humans; Introns; Nucleic Acid Conformation; RNA Splicing; RNA, Small Nuclear; Schizosaccharomyces; Spliceosomes
Biochemistry, Biophysics, and Structural Biology | Bioinformatics | Computational Biology | Integrative Biology | Systems Biology
Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2.U5.U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing.
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Citation: RNA. 2014 Mar;20(3):308-20. doi: 10.1261/rna.040980.113. Epub 2014 Jan 17. Link to article on publisher's site
RNA (New York, N.Y.)
Chen, Weijun; Shulha, Hennady P.; Ashar-Patel, Ami; Yang, Jing; Green, Karin M.; Query, Charles C.; Rhind, Nicholas R.; Weng, Zhiping; and Moore, Melissa J., "Endogenous U2.U5.U6 snRNA complexes in S. pombe are intron lariat spliceosomes" (2014). Program in Bioinformatics and Integrative Biology Publications and Presentations. 58.
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