A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.

The SEN) gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SENI gene and characterized the SENI mRNA, the SEN) gene product, the temperature-sensitive seni-) mutation, and three SENI null alleles. The SENI gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SENI, we detect a protein corresponding to the predicted molecular weight of SEN1. The SENI protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SENI protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPFI gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive seni-) allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

Mutations in three Escherichia coli K-12 genes were isolated that reduce the efficiency of the lysine-inserting nonsense suppressor supL. These antisuppressor mutations asuD, asuE, and asuF map at 61.9, 25.3, and 76.3 min, respectively, on the E. coli chromosome. Biochemical and genetic analysis of the mutant strains revealed the reason for the antisuppressor phenotype for two of these genes. The activity of lysyl-tRNA synthetase was reduced in strains with asuD mutations. The modification of 5-methylaminomethyl-2-thiouridine, the wobble base of tRNALYS, was impaired in asuE mutant strains, presumably at the 2-thiolation step. CGSCa J. Davies J. Miller CGSC CGSC CGSC J. Miller J. Miller J. Miller CGSC CGSC

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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