| Proteins that use the energy of NTP hydrolysis are thought to mediate the large number of conformational changes that are necessary for spliceosome assembly, activation, and recycling. Snu114, an essential and conserved U5 snRNP protein, is homologous to EF-G, the GTPase that induces translocation of tRNA on the ribosome. This raises the intriguing possibility that Snu114 may similarly couple the hydrolysis of GTP with conformational changes in the spliceosome. Based on the EF-G homology, domains III-V of Snu114 are predicted to undergo a large rearrangement following GTP hydrolysis.;To investigate Snu114 activity in Saccharomyces cerevisiae, I generated conditionally lethal alleles of the protein. The allele snu114-60, which truncates the 70 carboxy-terminal amino acids, is synthetically lethal with mutations in factors required for spliceosome activation, including the core U5 snRNP protein PRP8 and the ATPases PRP28 and BRR2, which release U1 and U4 from the spliceosome. When spliceosomes are assembled in vitro at the non-permissive temperature, snu114-60 blocks the release of U4 snRNA. We propose that activation of the spliceosome is dependent on GTP hydrolysis by Snu114, which leads to a rearrangement between the C-terminus of the protein and Prp8. This, in turn, would activate the ATPases Prp28 and Brr2.;A second class of SNU114 mutations greatly reduces the formation of the U5 snRNP and U5•U4/U6 tri-snRNPs. In these mutants, the interaction between Snu114 and Prp8 is reduced, and the total levels of Prp8 are decreased. These mutations fall within conserved motifs in the GTPase domain and in small clusters within each of domains III-V. We predict that these mutations either directly affect GTP binding/hydrolysis, or impact a rearrangement of the protein resulting from changes in nucleotide binding. We propose that Snu114 must bind GTP in order to interact productively with Prp8 and that this GTP-bound heterodimer is required in turn to assemble the U5 snRNP. This strategy would ensure that Snu114 is bound to GTP when it joins the spliceosome; subsequent GTP hydrolysis would then lead to spliceosome activation. |
