The ribosomal translocase homologue Snu114p is involved in unwinding U4/U6 RNA during activation of the spliceosome.
Snu114p is a yeast U5 snRNP protein homologous to the ribosomal elongation factor EF-2. Snu114p exhibits the same domain structure as EF-2, including the G-domain, but with an additional N-terminal domain. To test whether Snu114p in the spliceosome is involved in rearranging RNA secondary structures (by analogy to EF-2 in the ribosome), we created conditionally lethal mutants. Deletion of this N-terminal domain (snu114 Delta N) leads to a temperature-sensitive phenotype at 37 degrees C and a pre-mRNA splicing defect in vivo. Heat treatment of snu114 Delta N extracts blocked splicing in vitro before the first step. The snu114 Delta N still associates with the tri-snRNP, and the stability of this particle is not significantly impaired by thermal inactivation. Heat treatment of snu114 Delta N extracts resulted in accumulation of arrested spliceosomes in which the U4 RNA was not efficiently released, and we show that U4 is still base paired with the U6 RNA. This suggests that Snu114p is involved, directly or indirectly, in the U4/U6 unwinding, an essential step towards spliceosome activation.
Max-Planck-Institute of Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
Cornelia
C
Bartels
Christine
C
Klatt
Reinhard
R
Lührmann
Patrizia
P
Fabrizio
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D017353:Gene Deletion; D006358:Hot Temperature; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear; D012270:Ribosomes / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D012333:RNA, Messenger; 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