title: Human RNA 5'-kinase (hClp1) can function as a tRNA splicing enzyme in vivo.
abstract: Yeast and human Clp1 proteins are homologous components of the mRNA 3'-cleavage-polyadenylation machinery. Recent studies highlighting an association of human Clp1 (hClp1) with tRNA splicing endonuclease and an intrinsic RNA-specific 5'-OH polynucleotide kinase activity of hClp1 have prompted speculation that Clp1 might play a catalytic role in tRNA splicing in animal cells. Here, we show that expression of hClp1 in budding yeast can complement conditional and lethal mutations in the essential 5'-OH RNA kinase module of yeast or plant tRNA ligases. The tRNA splicing activity of hClp1 in yeast is abolished by mutations in the kinase active site. In contrast, overexpression of yeast Clp1 (yClp1) cannot rescue kinase-defective tRNA ligase mutants, and, unlike hClp1, the purified recombinant yClp1 protein has no detectable RNA kinase activity in vitro. Mutations of the yClp1 ATP-binding site do not affect yeast viability. These findings, and the fact that hClp1 cannot complement growth of a yeast clp1Delta strain, indicate that yeast and human Clp1 proteins are not functional orthologs, despite their structural similarity. Although hClp1 can perform the 5'-end-healing step of a yeast-type tRNA splicing pathway in vivo, it is uncertain whether its kinase activity is necessary for tRNA splicing in human cells, given that other mammalian counterparts of yeast-type tRNA repair enzymes are nonessential in vivo.
authors.affiliation: Graduate Program in Molecular Biology, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Ramirez
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
authors.forename: Beate
authors.initials: B
authors.lastname: Schwer
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018628:Gene Dosage; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000235:genetics / Q000378:metabolism; D011118:Polynucleotide Ligases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D012343:RNA, Transfer / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D009687:Nuclear Proteins; D014157:Transcription Factors; C059150:tRNA ligase, yeast; D012343:RNA, Transfer; C520030:CLP1 protein, human; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010727:Phosphoric Diester Hydrolases; D011118:Polynucleotide Ligases