Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease.
The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1+ gene as a multi-copy suppressor of snm1. The pac1+ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1+ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1+ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.
Rotondo
G
G
Department of Microbiology, New York University Medical Center, NY 10016, USA.
Gillespie
M
M
Frendewey
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D012342:RNA, Small Nuclear / Q000096:biosynthesis / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C090162:PSO2 protein, S cerevisiae; C499540:pac1 protein, S pombe