Functions of the yeast U6 RNA internal stem-loop during pre-messenger RNA splicing

The U6 spliceosomal RNA contains a highly conserved internal stem-loop (ISL) that must be unwound to allow its residues to base pair with the U4 RNA during spliceosome assembly. Upon activation of the spliceosome, this U4/U6 complex is unwound and the ISL is reformed in the catalytically active U2/U6 complex. The ISL contains an internal loop region that coordinates a Mg++ ion essential for the first catalytic step of splicing. This thesis examines U6 ISL function during pre-mRNA splicing.;Directed genetic analyses were used to determine the cause(s) of growth defects resulting from mutations in Saccharomyces cerevisiae U6 ISL internal loop positions 67, 79, and 80. The lethality of mutations C67A and C67G results from a disruption of base pairing potential between U4 and U6, as these mutations are fully suppressed both by U4 overexpression and by U4 mutations that restore pairing potential. C67A and C67G are also suppressed by mutations in U6 snRNP protein Prp24 that were previously shown to suppress defects in U4/U6 pairing. Suppressors of U80G lethality have a severe heat-sensitive growth defect that does not result from a block in U4/U6 assembly. I provide evidence that the U6 ISL internal loop functions in U4/U6 assembly and at least one additional process in the splicing cycle. Genetic interactions between the ISL internal loop and the U6 snRNP protein Prp24 imply a role for the internal loop in spliceosome activation.;To further study ISL function, splicing extracts were prepared from the U80G suppressor strain U4/U6-Trip1. These extracts are defective for pre-mRNA splicing at elevated temperatures, and assemble spliceosomes that exhibit aberrant native gel migration, suggesting conformational or compositional alterations. Analysis of affinity-purified U4/U6-Trip I spliceosomes revealed elevated levels of U1 snRNP even at splicing permissive temperature and a defect in U4/U6 unwinding at restrictive temperature. These results implicate the ISL internal loop in a novel function, the coordination of U1 snRNP release and U4/U6 unwinding during spliceosome activation.