title: Saccharomyces cerevisiae Ngl3p is an active 3'-5' exonuclease with a specificity towards poly-A RNA reminiscent of cellular deadenylases.
abstract: Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes.
authors.affiliation: Department of Molecular Biology and Genetics, Centre for mRNP Biogenesis and Metabolism, Aarhus University, Gustav Wieds Vej 10c and C. F. Møllers Allé 130, DK-8000 Aarhus C, Denmark.
authors.forename: Ane
authors.initials: A
authors.lastname: Feddersen
authors.forename: Emil
authors.initials: E
authors.lastname: Dedic
authors.forename: Esben G
authors.initials: EG
authors.lastname: Poulsen
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmid
authors.forename: Lan Bich
authors.initials: LB
authors.lastname: Van
authors.forename: Torben Heick
authors.initials: TH
authors.lastname: Jensen
authors.forename: Ditlev E
authors.initials: DE
authors.lastname: Brodersen
mesh_terms: D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000378:metabolism*; D011068:Poly G / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D011061:Poly A; D011068:Poly G; D005095:Exoribonucleases; C570638:Ngl3 protein, S cerevisiae; D012260:Ribonucleases; C445084:mRNA deadenylase