Abstract
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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Acknowledgements
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).
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A.D., supervised by B.S., designed and performed the majority of the experiments. E.L., with E.C., performed the initial sequence alignment for the subunits with similarity to archaeal phosphorolytic proteins and carried out expression of all recombinant proteins. A.D., E.L., E.C. and B.S. wrote the paper.
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Supplementary information
Supplementary Fig. 1
Sequence alignment. (PDF 169 kb)
Supplementary Fig. 2
Nuclease assay. (PDF 727 kb)
Supplementary Fig. 3
dis3 growth phenotype. (PDF 340 kb)
Supplementary Fig. 4
rrp41 growth phenotype. (PDF 389 kb)
Supplementary Fig. 5
mRNA decay graph. (PDF 454 kb)
Supplementary Fig. 6
Dis3 activity. (PDF 232 kb)
Supplementary Table 1
Yeast strains. (PDF 77 kb)
Supplementary Table 2
Oligonucleotides. (PDF 49 kb)
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Dziembowski, A., Lorentzen, E., Conti, E. et al. A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nat Struct Mol Biol 14, 15–22 (2007). https://doi.org/10.1038/nsmb1184
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DOI: https://doi.org/10.1038/nsmb1184
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