Mutagenesis suggests several roles of Snu114p in pre-mRNA splicing.
Snu114p, a yeast U5 small nuclear ribonucleoprotein (snRNP) homologous to the ribosomal GTPase EF-2, was recently found to play a part in the dissociation of U4 small nuclear RNA (snRNA) from U6 snRNA. Here, we show that purified Snu114p binds GTP specifically. To test the possibility that binding and hydrolysis of GTP by Snu114p are required to stimulate the unwinding of U4 from U6, we produced several mutations of Snu114p. Residues whose mutations led to lethal phenotypes were all clustered in the P loop and in the guanine-ring binding sequence (NKXD) of the G domain, which in elongation factor-G is required for the binding and hydrolysis of GTP. An arginine residue in domain II, which in EF-G forms a salt bridge with a residue of the G domain, when mutated in Snu114p (R487E), led to a temperature-sensitive phenotype. The substitution D271N in the NKXD sequence is predicted to bind XTP instead of GTP. Spliceosomes containing this mutant, isolated by affinity chromatography after heat treatment, retained U4 snRNA paired with the U6 snRNA. U4 snRNA was released efficiently only when these arrested spliceosomes were reactivated by lowering the temperature in the presence of a mixture of ATP and XTP. Because non-hydrolyzable XTP analogues did not consent the release of U4, we conclude that the release requires hydrolysis of XTP. This suggests that Snu114p needs GTP to influence, directly or indirectly, the unwinding of U4 from U6. An additional role for Snu114p is also demonstrated: after growth of the D271N and R487E strains at high temperatures, we observed decreased levels of the U5 and the U4/U6.U5 snRNPs. This indicates that, before splicing, Snu114p plays a part in the assembly of both particles.
Max-Planck-Institute of Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Cornelia
C
Bartels
Henning
H
Urlaub
Reinhard
R
Luhrmann
Patrizia
P
Fabrizio
D000595:Amino Acid Sequence; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D006147:Guanine / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism; D013696:Temperature
D003432:Cross-Linking Reagents; D012342:RNA, Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; C472240:SNU114 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA; D006147:Guanine; D012313:RNA; D006160:Guanosine Triphosphate; D005990:Glycerol