The invariant U5 snRNA loop 1 sequence is dispensable for the first catalytic step of pre-mRNA splicing in yeast.

We have developed an in vitro reconstitution system to investigate the role of U5 snRNA in the two catalytic steps of pre-mRNA splicing. The invariant U5 loop 1 is known to interact with exon sequences at the 5' splice site before the first catalytic step. Remarkably, analysis of U5 mutations in vitro reveals that the first transesterification occurs accurately in the absence of the U5 loop. Therefore this sequence is not an essential component of the spliceosomal active site for the first catalytic step. The second catalytic step, although strongly dependent on the presence of a U5 loop to tether the exon 1 splicing intermediate, is surprisingly tolerant of mutations in the invariant sequence.