SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome.
Pre-spliceosomes, formed in HeLa nuclear extracts and isolated by sedimentation on glycerol gradients, were chased into spliceosomes, the macromolecular enzyme that catalyzes intron removal. We demonstrate that the pre-spliceosome to spliceosome transition was dependent on ATP hydrolysis and required both a U-rich small nuclear ribonucleoprotein (U snRNP)-containing fraction and a fraction of non-snRNP factors. The active components in the non-snRNP fraction were identified as SR proteins and were purified to apparent homogeneity. Recombinant SR proteins (ASF, SC35, SRp55), as well as gel-purified SR proteins, with the exception of SRp20, were able to restore efficient spliceosome formation. We also demonstrate that the pre-spliceosome to spliceosome transition requires phosphorylated SR proteins. This is the first evidence that SR proteins are required for the pre-spliceosome to spliceosome transition, the step at which the U4/U6.U5 tri-snRNP assembles on the pre-mRNA. The results shown here, together with previous data, suggest U snRNPs require SR proteins as escorts to enter the assembling spliceosome.
Roscigno
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RF
Department of Molecular Cancer Biology, Levine Science Research Center, Duke University Medical Center, Durham, North Carolina 27710, USA.
Garcia-Blanco
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MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D002458:Cell Fractionation; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000378:metabolism*
D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D000255:Adenosine Triphosphate