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The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.

Schneider C, Leung E, Brown J, Tollervey D - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.

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.

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The rrp44-exo mutation is not synthetically lethal with deletion of Rrp6. (A) and (B). In vivo analyses of yeast strains expressing Rrp44-exo in the presence or absence of the exosome components Rrp6 or Rrp41. RNA was separated on an 8% polyacrylamide/8 M urea gel (A) or an 1% agarose/glyoxal gel (B) and analyzed as described in Figure 1D.
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Figure 3: The rrp44-exo mutation is not synthetically lethal with deletion of Rrp6. (A) and (B). In vivo analyses of yeast strains expressing Rrp44-exo in the presence or absence of the exosome components Rrp6 or Rrp41. RNA was separated on an 8% polyacrylamide/8 M urea gel (A) or an 1% agarose/glyoxal gel (B) and analyzed as described in Figure 1D.

Mentions: ‘Since Rrp44 is essential for viability, whereas its exonuclease activity is largely dispensable, it seemed likely that it was partially redundant with the activities of one or more other nucleases. An obvious possibility was the nuclear, exosome-associated exonuclease Rrp6. Indeed, the rrp44-exo mutation was reported to result in synthetic lethality with an rrp6Δ (4). However, this was not observed in our strains (Figures 2 and 3). To test for synthetic lethality between rrp44-exo and rrp6Δ, the RRP6 ORF was first deleted in the rrp44Δ strain complemented with a plasmid carrying RRP44 and a URA3 selective marker (see above). A second plasmid (pRS315) carrying LEU2 and expressing either Rrp44 or Rrp44-exo was subsequently introduced and the URA3 plasmid was then counter-selected on 5-FOA containing SD -His/-Leu plates. The growth of individual transformants was then analyzed on SD -His/-Leu medium at 25°C (Figure 2). The loss of Rrp44-exonuclease activity reduced the growth of RRP6 and rrp6Δ by a similar proportion, relative to the same strains expressing Rrp44 (Figure 2).


The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.

Schneider C, Leung E, Brown J, Tollervey D - Nucleic Acids Res. (2009)

The rrp44-exo mutation is not synthetically lethal with deletion of Rrp6. (A) and (B). In vivo analyses of yeast strains expressing Rrp44-exo in the presence or absence of the exosome components Rrp6 or Rrp41. RNA was separated on an 8% polyacrylamide/8 M urea gel (A) or an 1% agarose/glyoxal gel (B) and analyzed as described in Figure 1D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651783&req=5

Figure 3: The rrp44-exo mutation is not synthetically lethal with deletion of Rrp6. (A) and (B). In vivo analyses of yeast strains expressing Rrp44-exo in the presence or absence of the exosome components Rrp6 or Rrp41. RNA was separated on an 8% polyacrylamide/8 M urea gel (A) or an 1% agarose/glyoxal gel (B) and analyzed as described in Figure 1D.
Mentions: ‘Since Rrp44 is essential for viability, whereas its exonuclease activity is largely dispensable, it seemed likely that it was partially redundant with the activities of one or more other nucleases. An obvious possibility was the nuclear, exosome-associated exonuclease Rrp6. Indeed, the rrp44-exo mutation was reported to result in synthetic lethality with an rrp6Δ (4). However, this was not observed in our strains (Figures 2 and 3). To test for synthetic lethality between rrp44-exo and rrp6Δ, the RRP6 ORF was first deleted in the rrp44Δ strain complemented with a plasmid carrying RRP44 and a URA3 selective marker (see above). A second plasmid (pRS315) carrying LEU2 and expressing either Rrp44 or Rrp44-exo was subsequently introduced and the URA3 plasmid was then counter-selected on 5-FOA containing SD -His/-Leu plates. The growth of individual transformants was then analyzed on SD -His/-Leu medium at 25°C (Figure 2). The loss of Rrp44-exonuclease activity reduced the growth of RRP6 and rrp6Δ by a similar proportion, relative to the same strains expressing Rrp44 (Figure 2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.

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.

Show MeSH
Related in: MedlinePlus