An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.
We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).
D002846:Chromatography, Affinity / Q000379:methods; D007700:Kinetics; D010957:Plasmids; D012322:RNA Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012322:RNA Precursors; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear