The conserved core of the exosome, the major eukaryotic 3′ → 5′ exonuclease, contains nine subunits that form a ring similar to the phosphorolytic bacterial PNPase and archaeal exosome, as well as Dis3. Dis3 is homologous to bacterial RNase II, a hydrolytic enzyme. Previous studies have suggested that all subunits are active 3′ → 5′ exoRNases. We show here that Dis3 is responsible for exosome core activity. The purified exosome core has a hydrolytic, processive and Mg2+-dependent activity with characteristics similar to those of recombinant Dis3. Moreover, a catalytically inactive Dis3 mutant has no exosome core activity in vitro and shows in vivo RNA degradation phenotypes similar to those resulting from exosome depletion. In contrast, mutations in Rrp41, the only subunit carrying a conserved phosphorolytic site, appear phenotypically not different from wild-type yeast. We observed that the yeast exosome ring mediates interactions with protein partners, providing an explanation for its essential function.
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We acknowledge C. Faux for constructing the dcp1-2 mutant strain, C. Henry (PAPSS, INRA, Jouy-en-Josas) for mass spectrometry analysis and the Unité Pilote (ICSN, Gif-sur-Yvette) for fermentation service. We are grateful to group members for useful discussions and S. Camier-Thuillier, A. Lebreton and F. Mauxion for manuscript corrections. A.D. was supported by fellowships from the Foundation for Polish Science and the Human Frontier Science Program. This work was supported by La Ligue contre le Cancer (Equipe Labellisée 2005), the Human Frontier Science Program, the Agence Nationale de la Recherche (ANR-05-BLAN-0062-03), Centre National de la Recherche Scientifique and the EU Sixth Framework Program 3D-Repertoire project (512028).
The authors declare no competing financial interests.
Sequence alignment. (PDF 169 kb)
Nuclease assay. (PDF 727 kb)
dis3 growth phenotype. (PDF 340 kb)
rrp41 growth phenotype. (PDF 389 kb)
mRNA decay graph. (PDF 454 kb)
Dis3 activity. (PDF 232 kb)
Yeast strains. (PDF 77 kb)
Oligonucleotides. (PDF 49 kb)
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