title: Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity.
abstract: Removal of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20-30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min(-1) and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Martin
authors.forename: José
authors.initials: J
authors.lastname: Rino
authors.forename: Célia
authors.initials: C
authors.lastname: Carvalho
authors.forename: Tomas
authors.initials: T
authors.lastname: Kirchhausen
authors.forename: Maria
authors.initials: M
authors.lastname: Carmo-Fonseca
mesh_terms: D000818:Animals; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D051379:Mice; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D055544:beta-Globins / Q000235:genetics
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; D055544:beta-Globins
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