Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences.
We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000260:Adenoviruses, Human; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D007700:Kinetics; D009154:Mutation; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D012322:RNA Precursors; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D000255:Adenosine Triphosphate