title: The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.
abstract: Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3' exonuclease activity. We report that expression only of Rrp44 lacking 3'-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5'-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3'-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Schneider
authors.forename: Eileen
authors.initials: E
authors.lastname: Leung
authors.forename: Jeremy
authors.initials: J
authors.lastname: Brown
authors.forename: David
authors.initials: D
authors.lastname: Tollervey
mesh_terms: D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D005095:Exoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D063326:Exosome Multienzyme Ribonuclease Complex; D017353:Gene Deletion; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D004722:Endoribonucleases; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C068398:XRN1 protein, S cerevisiae; C542283:DIS3 protein, S cerevisiae; C112610:RRP6 protein, S cerevisiae