Genetic Analysis Reveals a Role for the C Terminus of the Saccharomyces cerevisiae GTPase Snu114 During Spliceosome Activation

Abstract

Abstract Snu114 is the only GTPase required for mRNA splicing. As a homolog of elongation factor G, it contains three domains (III–V) predicted to undergo a large rearrangement following GTP hydrolysis. To assess the functional importance of the domains of Snu114, we used random mutagenesis to create conditionally lethal alleles. We identified three main classes: (1) mutations that are predicted to affect GTP binding and hydrolysis, (2) mutations that are clustered in 10- to 20-amino-acid stretches in each of domains III–V, and (3) mutations that result in deletion of up to 70 amino acids from the C terminus. Representative mutations from each of these classes blocked the first step of splicing in vivo and in vitro. The growth defects caused by most alleles were synthetically exacerbated by mutations in PRP8, a U5 snRNP protein that physically interacts with Snu114, as well as in genes involved in snRNP biogenesis, including SAD1 and BRR1. The allele snu114-60, which truncates the C terminus, was synthetically lethal with factors required for activation of the spliceosome, including the DExD/H-box ATPases BRR2 and PRP28. We propose that GTP hydrolysis results in a rearrangement between Prp8 and the C terminus of Snu114 that leads to release of U1 and U4, thus activating the spliceosome for catalysis.