The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.
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.
Schneider
Claudia
C
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.
Leung
Eileen
E
Brown
Jeremy
J
Tollervey
David
D
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*
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