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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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Growth analysis of yeast strains expressing Rrp44-exo. Yeast strains carrying either rrp44Δ or rrp44Δ rrp6Δ were transformed with plasmids expressing WT Rrp44 (WT) or Rrp44 with the D551N mutation (exo). To analyze growth, strains were grown in selective liquid glucose medium at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ), which are their respective optima.
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Figure 2: Growth analysis of yeast strains expressing Rrp44-exo. Yeast strains carrying either rrp44Δ or rrp44Δ rrp6Δ were transformed with plasmids expressing WT Rrp44 (WT) or Rrp44 with the D551N mutation (exo). To analyze growth, strains were grown in selective liquid glucose medium at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ), which are their respective optima.

Mentions: Constructs expressed under the GAL promoter are never fully repressed, and so a low level of Rrp44 will always be expressed in a GAL::rrp44 strain. To avoid this problem, the RRP44 ORF was precisely deleted in strain BY4741 supplemented with a plasmid carrying RRP44 and a URA3 selective marker. Following construction of rrp44Δ, a second plasmid was introduced that carried LEU2 and expressed either Rrp44 or Rrp44-exo. Mitotic segregants that had lost the URA3 plasmid containing wild type RRP44 were then isolated on plates containing 5-FOA, which selectively kills cells expressing Ura3. The rrp44Δ strains showed an increased doubling time when complemented by expression of Rrp44-exo (3 h when compared to 2 h for WT-Rrp44, Figure 2), similar to the results obtained with GAL::rrp44 strains (Figure 1B).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)

Growth analysis of yeast strains expressing Rrp44-exo. Yeast strains carrying either rrp44Δ or rrp44Δ rrp6Δ were transformed with plasmids expressing WT Rrp44 (WT) or Rrp44 with the D551N mutation (exo). To analyze growth, strains were grown in selective liquid glucose medium at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ), which are their respective optima.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Growth analysis of yeast strains expressing Rrp44-exo. Yeast strains carrying either rrp44Δ or rrp44Δ rrp6Δ were transformed with plasmids expressing WT Rrp44 (WT) or Rrp44 with the D551N mutation (exo). To analyze growth, strains were grown in selective liquid glucose medium at 30°C (rrp44Δ) or 25°C (rrp44Δ rrp6Δ), which are their respective optima.
Mentions: Constructs expressed under the GAL promoter are never fully repressed, and so a low level of Rrp44 will always be expressed in a GAL::rrp44 strain. To avoid this problem, the RRP44 ORF was precisely deleted in strain BY4741 supplemented with a plasmid carrying RRP44 and a URA3 selective marker. Following construction of rrp44Δ, a second plasmid was introduced that carried LEU2 and expressed either Rrp44 or Rrp44-exo. Mitotic segregants that had lost the URA3 plasmid containing wild type RRP44 were then isolated on plates containing 5-FOA, which selectively kills cells expressing Ura3. The rrp44Δ strains showed an increased doubling time when complemented by expression of Rrp44-exo (3 h when compared to 2 h for WT-Rrp44, Figure 2), similar to the results obtained with GAL::rrp44 strains (Figure 1B).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