Endonucleolytic RNA cleavage by a eukaryotic exosome

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Abstract
The exosome is a multisubunit exonuclease complex that degrades many types of RNAs, in many different contexts, in a 3' to 5' manner. The catalytic component of the exosome is the Dis3 subunit. Dis3 contains a PIN domain, which is sometimes associated with nuclease activity. In this work, Lebreton et al. show that the Dis3 PIN domain possesses endonuclease activity (that is, it can cleave RNA internally, rather than from an end). Mutations in either this domain or in the exonuclease domain exhibit a growth phenotype, suggesting that both activities are physiologically important. While other RNase complexes contain both endo- and exonuclease activities, this is the first example of both activities being encoded in a single protein. The exosome is a multisubunit exonuclease complex that degrades many types of RNAs, in many different contexts, in a 3′ to 5′ manner. The catalytic component of the exosome is the Dis3 subunit. Dis3 contains a PIN domain, which is sometimes associated with nuclease activity. This work shows that the Dis3 PIN domain also possesses endonuclease activity (that is, it can cleave RNA internally, rather than from an end). Mutations in either this domain or in the exonuclease domain exhibit a growth phenotype, suggesting that both activities are physiologically important. 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.