Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA.
Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.
Dobbyn
Helen C
HC
School of Biological Sciences, University of Manchester, Manchester, M13 9PT, UK.
O'Keefe
Raymond T
RT
D000595:Amino Acid Sequence; D005690:Galactose / Q000378:metabolism; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D009154:Mutation; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D012342:RNA, Small Nuclear / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
D012322:RNA Precursors; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; C117378:RNA, U3 small nucleolar; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C440569:Snu13 protein, S cerevisiae; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA; D005690:Galactose