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

Cyrille Girard, Cindy L. Will, Jianhe Peng, Evgeny M. Makarov, Berthold Kastner, Ira Lemm, Henning Urlaub, Klaus Hartmuth () and Reinhard Lührmann ()
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Cyrille Girard: Max Planck Institute for Biophysical Chemistry
Cindy L. Will: Max Planck Institute for Biophysical Chemistry
Jianhe Peng: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry
Evgeny M. Makarov: Max Planck Institute for Biophysical Chemistry
Berthold Kastner: Max Planck Institute for Biophysical Chemistry
Ira Lemm: Max Planck Institute for Biophysical Chemistry
Henning Urlaub: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry
Klaus Hartmuth: Max Planck Institute for Biophysical Chemistry
Reinhard Lührmann: Max Planck Institute for Biophysical Chemistry

Nature Communications, 2012, vol. 3, issue 1, 1-12

Abstract: 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.

Date: 2012
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DOI: 10.1038/ncomms1998

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