SEN1, a Positive Effector of tRNA-Splicing Endonuclease in Saccharomyces cerevisiae

Abstract

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.

The sen1+ Gene of Schizosaccharomyces pombe, a Homologue of Budding Yeast SEN1, Encodes an RNA and DNA Helicase

“…We have isolated two enzymes from extracts of Schizosaccharomyces pombe that were purified on the basis of their ATPase activity that was stimulated by either poly(U) or M13 sscDNA. Peptide sequence analyses of the purified proteins confirmed that the enzymes were encoded by the S. pombe homologue of SEN1 (splicing endonuclease), originally reported to be involved in the tRNA splicing pathway in S. cereVisiae (17,18). The removal of introns from precursor tRNAs is mediated by three catalytic enzymes: a site-specific tetrameric endonuclease that catalyzes both 5′ and 3′ cleavages (18)(19)(20), a monomeric tRNA ligase (21,22), and an NAD-dependent phosphotransferase that removes a 2′phosphate from the splice junction to produce mature tRNA (23).…”

“…Peptide sequence analyses of the purified proteins confirmed that the enzymes were encoded by the S. pombe homologue of SEN1 (splicing endonuclease), originally reported to be involved in the tRNA splicing pathway in S. cereVisiae (17,18). The removal of introns from precursor tRNAs is mediated by three catalytic enzymes: a site-specific tetrameric endonuclease that catalyzes both 5′ and 3′ cleavages (18)(19)(20), a monomeric tRNA ligase (21,22), and an NAD-dependent phosphotransferase that removes a 2′phosphate from the splice junction to produce mature tRNA (23). Whereas each of these proteins provides a unique enzymatic activity in the precursor-tRNA splicing pathway, the role played by ScSen1p is not clearly understood at present.…”

De novo pathogenic variant in SETX causes a rapidly progressive neurodegenerative disorder of early childhood-onset with severe axonal polyneuropathy

“…Strong expression within the neuromuscular axis results from use of this promoter. Given that SETX protein is maintained at tightly controlled levels [ 6 , 13 ], total protein levels were not elevated for combined mouse Setx and human SETX. Specifically, Arg2136His mutant SETX was determined to represent ~ 71% of total SETX protein in transgenic line #1920 that was used in this study.…”

A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence

“…Translational fusions were made between the two elements and a NLS-lacZ fusion contained on pDU254 (Ursic et al, 1995). The NLS in this fusion is from yeast SEN1, which codes for a putative nuclear RNA helicase (DeMarini et al, 1992). Complementary oligonucleotides (LSO103-LSO106, Table 2) were synthesized containing the UPF3 NES-A and NES-B sequences.…”

“…7A). The two putative NES elements identified in Upf3p R. L. Shirley and others In order to test whether NES-A and NES-B can function independently as signals for nuclear export, the NES elements were fused to a reporter protein consisting of a NLS derived from the yeast Sen1p protein (DeMarini et al, 1992) and βgalactosidase (see Materials and Methods). The NLS in Sen1p causes the wild-type protein and reporter fusions containing the NLS to localize exclusively to the nucleus with no staining in the cytoplasm (Ursic et al, 1995).…”