title: Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion.
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.
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077                 Göttingen, Germany.
authors.forename: Cyrille
authors.initials: C
authors.lastname: Girard
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.forename: Jianhe
authors.initials: J
authors.lastname: Peng
authors.forename: Evgeny M
authors.initials: EM
authors.lastname: Makarov
authors.forename: Berthold
authors.initials: B
authors.lastname: Kastner
authors.forename: Ira
authors.initials: I
authors.lastname: Lemm
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Klaus
authors.initials: K
authors.lastname: Hartmuth
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology*; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human