The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

J Cell Biol. 2010 Oct 4;191(1):75-86. doi: 10.1083/jcb.201004030.

Abstract

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Nucleus / metabolism
  • Fluorescence Recovery After Photobleaching
  • HeLa Cells
  • Humans
  • Kinetics
  • RNA Precursors / metabolism*
  • RNA Splicing / physiology*
  • RNA, Messenger / metabolism*
  • Ribonucleoproteins, Small Nuclear / metabolism*
  • Ribonucleoproteins, Small Nuclear / physiology
  • Spectrometry, Fluorescence
  • Spliceosomes / metabolism*

Substances

  • RNA Precursors
  • RNA, Messenger
  • Ribonucleoproteins, Small Nuclear