Mutations in U5 snRNA loop 1 influence the splicing of different genes in vivo.
The U5 snRNA loop 1 is characterized by the conserved sequence G1C2C3U4U5U6Y7A8Y9 and is essential for the alignment of exons during the second step of pre-mRNA splicing in Saccharo myces cerevisiae. Despite this sequence conservation the size, rather than sequence, of loop 1 is critical for exon alignment in vitro. To determine the in vivo requirements for U5 loop 1 a library of loop 1 sequences was transformed into a yeast strain where the endogenous U5 gene was deleted. Comparison of viable mutations in loop 1 revealed that position 6 was invariant and positions 5 and 7 displayed some sequence conservation. These data indicate positions 5, 6 and 7 in loop 1 are important for U5 function in vivo. A screen for mutations that suppress the temperature-sensitive phenotype of three loop 1 mutants produced eight intragenic suppressors all containing alterations in loop 1. Further analysis of these temperature-sensitive mutants revealed that each displayed distinct cell cycle arrest phenotypes and pre-mRNA splicing inhibition patterns. The cell cycle arrest is likely attributed to inefficient splicing of alpha-tubulin pre-mRNA in one mutant and actin pre-mRNA in another. These results suggest that various mutations in loop 1 may affect the splicing of different pre-mRNAs in vivo.
O'Keefe
Raymond T
RT
School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. rokeefe@man.ac.uk
D017398:Alternative Splicing; D001542:Benomyl / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics*; D009154:Mutation; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D013012:Sorbitol / Q000494:pharmacology; D013696:Temperature
D012322:RNA Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; C405485:U5 small nuclear RNA; D013012:Sorbitol; D001542:Benomyl