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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • HeLa Cells
  • Humans
  • Introns*
  • Mice
  • RNA Splice Sites / genetics
  • RNA Splicing*
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • beta-Globins / genetics


  • RNA Splice Sites
  • RNA, Messenger
  • beta-Globins