Single-molecule imaging of transcriptionally coupled and uncoupled splicing

Diana Y. Vargas, Khyati Shah, Mona Batish, Michael Levandoski, Sourav Sinha, Salvatore A.E. Marras, Paul Schedl, Sanjay Tyagi

Research output: Contribution to journalArticlepeer-review

148 Scopus citations

Abstract

Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA-binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, whereas the preceding and succeeding introns are removed cotranscriptionally.

Original languageEnglish (US)
Pages (from-to)1054-1065
Number of pages12
JournalCell
Volume147
Issue number5
DOIs
StatePublished - Nov 23 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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