Assembly of Snu114 into U5 snRNP requires Prp8 and a functional GTPase domain.
Snu114 is a U5 snRNP protein essential for pre-mRNA splicing. Based on its homology with the ribosomal translocase EF-G, it is thought that GTP hydrolysis by Snu114 induces conformational rearrangements in the spliceosome. We recently identified allele-specific genetic interactions between SNU114 and genes encoding three other U5 snRNP components, Prp8 and two RNA-dependent ATPases, Prp28 and Brr2, required for destabilization of U1 and U4 snRNPs prior to catalysis. To shed more light onto the function of Snu114, we have now directly analyzed snRNP and spliceosome assembly in SNU114 mutant extracts. The Snu114-60 C-terminal truncation mutant, which is synthetically lethal with the ATPase mutants prp28-1 and brr2-1, assembles spliceosomes but subsequently blocks U4 snRNP release. Conversely, mutants in the GTPase domain fail to assemble U5 snRNPs. These mutations prevent the interaction of Snu114 with Prp8 as well as with U5 snRNA. Since Prp8 is thought to regulate the activity of the DEAD-box ATPases, this strategy of snRNP assembly could ensure that Prp8 activity is itself regulated by a GTP-dependent mechanism.
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-2200, USA.
Tamara J
TJ
Brenner
Christine
C
Guthrie
D019921:Biotinylation; D005656:Fungal Proteins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D005656:Fungal Proteins; C077018:PRP8 protein, S cerevisiae; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; C472240:SNU114 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases