Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly.
Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.
Staknis
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Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
Reed
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D000818:Animals; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008322:Mammals; D008954:Models, Biological; D012322:RNA Precursors / Q000302:isolation & purification / Q000378:metabolism*; D012326:RNA Splicing; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000302:isolation & purification / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism* / Q000528:radiation effects / Q000648:ultrastructure; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D046911:Macromolecular Substances; D012322:RNA Precursors; D017413:Ribonucleoprotein, U2 Small Nuclear