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Endonucleolytic RNA cleavage by a eukaryotic exosome

Abstract

The exosome is a major eukaryotic nuclease located in both the nucleus and the cytoplasm that contributes to the processing, quality control and/or turnover of a large number of cellular RNAs1,2,3,4,5,6. This large macromolecular assembly has been described as a 3′→5′ exonuclease1 and shown to contain a nine-subunit ring structure evolutionarily related to archaeal exosome-like complexes and bacterial polynucleotide phosphorylases. Recent results have shown that, unlike its prokaryotic counterparts, the yeast and human ring structures are catalytically inactive. In contrast, the exonucleolytic activity of the yeast exosome core was shown to be mediated by the RNB domain of the eukaryote-specific Dis3 subunit7,8,9. Here we show, using in vitro assays, that yeast Dis3 has an additional endoribonuclease activity mediated by the PIN domain located at the amino terminus of this multidomain protein. Simultaneous inactivation of the endonucleolytic and exonucleolytic activities of the exosome core generates a synthetic growth phenotype in vivo, supporting a physiological function for the PIN domain. This activity is responsible for the cleavage of some natural exosome substrates, independently of exonucleolytic degradation. In contrast with current models, our results show that eukaryotic exosome cores have both endonucleolytic and exonucleolytic activities, mediated by two distinct domains of the Dis3 subunit. The mode of action of eukaryotic exosome cores in RNA processing and degradation should be reconsidered, taking into account the cooperation between its multiple ribonucleolytic activities.

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Figure 1: Endonucleolytic activity of the Dis3 PIN domain in vitro.
Figure 2: Synthetic cell-growth phenotypes of PIN mutants.
Figure 3: Dis3 PIN domain mediates cleavage of natural exosome substrates in vivo.

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Acknowledgements

We thank E. Conti and J. Basquin for providing the Dis3 PIN domain expression construct and 6×His-SUMO protease, and E. Conti, E. Lorentzen, J. Kufel and members of our groups for insightful discussions. This work was supported by La Ligue contre le Cancer (Équipe Labellisée 2008), Agence Nationale de la Recherche project CUTs, CNRS, ESF RNA Quality program (project EUxosome) and the FP6 EU grant 3D repertoire, and an EMBO installation grant. R.T. is the recipient of the Stipend for Young Researchers from the Foundation for Polish Science and was supported through a Faculty of Biology, University of Warsaw intramural grant.

Author Contributions R.T. expressed and purified the recombinant proteins and performed all the in vitro experiments under the supervision of A.D. A.L. performed all in vivo experiments under the supervision of B.S. All authors discussed the results and wrote the paper.

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Correspondence to Andrzej Dziembowski or Bertrand Séraphin.

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Lebreton, A., Tomecki, R., Dziembowski, A. et al. Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature 456, 993–996 (2008). https://doi.org/10.1038/nature07480

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