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Cell Rep. 2013 Sep 26;4(6):1144-55. doi: 10.1016/j.celrep.2013.08.013. Epub 2013 Sep 12.

Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity.

Author information

1
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.

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.

PMID:
24035393
PMCID:
PMC3805459
DOI:
10.1016/j.celrep.2013.08.013
[Indexed for MEDLINE]
Free PMC Article

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