A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly.
Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.
D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007163:Immunosorbent Techniques; D008836:Micrococcal Nuclease / Q000378:metabolism; D009994:Osmolar Concentration; D011189:Potassium Chloride; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
D002352:Carrier Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D011189:Potassium Chloride; D008836:Micrococcal Nuclease