Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion

Nat Commun. 2012;3:994. doi: 10.1038/ncomms1998.

Abstract

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism*
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation / genetics
  • Phosphorylation / physiology
  • RNA Splicing / genetics
  • RNA Splicing / physiology*
  • RNA Splicing Factors
  • Ribonucleoprotein, U2 Small Nuclear / genetics
  • Ribonucleoprotein, U2 Small Nuclear / metabolism
  • Spliceosomes / metabolism*

Substances

  • Phosphoproteins
  • RNA Splicing Factors
  • Ribonucleoprotein, U2 Small Nuclear
  • SF3B1 protein, human