title: The nucleic acid-binding domain and translational repression activity of a Xenopus terminal uridylyl transferase.
abstract: Terminal uridylyl transferases (TUTs) catalyze the addition of uridines to the 3' ends of RNAs and are implicated in the regulation of both messenger RNAs and microRNAs. To better understand how TUTs add uridines to RNAs, we focused on a putative TUT from Xenopus laevis, XTUT7. We determined that XTUT7 catalyzed the addition of uridines to RNAs. Mutational analysis revealed that a truncated XTUT7 enzyme, which contained solely the nucleotidyl transferase and poly(A) polymerase-associated domains, was sufficient for catalytic activity. XTUT7 activity decreased upon removal of the CCHC zinc finger domains and a short segment of basic amino acids (the basic region). This basic region bound nucleic acids in vitro. We also demonstrated that XTUT7 repressed translation of a polyadenylated RNA, to which it added a distinct number of uridines. We generated a predicted structure of the XTUT7 catalytic core that indicated histidine 1269 was likely important for uridine specificity. Indeed, mutation of histidine 1269 broadened the nucleotide specificity of XTUT7 and abolished XTUT7-dependent translational repression. Our data reveal key aspects of how XTUT7 adds uridines to RNAs, highlight the role of the basic region, illustrate that XTUT7 can repress translation, and identify an amino acid important for uridine specificity.
authors.affiliation: Integrated Program in Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Lapointe
authors.forename: Marvin
authors.initials: M
authors.lastname: Wickens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D005786:Gene Expression Regulation; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014529:Uridine / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
chemical_list: D009696:Nucleic Acids; D029867:Xenopus Proteins; D006639:Histidine; D012313:RNA; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C583621:TUT7 protein, Xenopus; C031687:UTP-RNA uridylyltransferase; D014529:Uridine
keywords: Enzyme Mutation; Molecular Biology; Protein-Nucleic Acid Interaction; RNA Uridylation; RNA-binding Protein; TUT7; Terminal Uridylyl Transferase; Translation Control; Xenopus; ZCCHC6