title: The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro.
abstract: The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.
authors: Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
authors: Karolina
authors: K
authors: Drazkowska
authors: Rafal
authors: R
authors: Tomecki
authors: Krystian
authors: K
authors: Stodus
authors: Katarzyna
authors: K
authors: Kowalska
authors: Mariusz
authors: M
authors: Czarnocki-Cieciura
authors: Andrzej
authors: A
authors: Dziembowski
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D002597:Chaetomium / Q000201:enzymology; D003593:Cytoplasm / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex / Q000235:genetics / Q000378:metabolism*; D005804:Genes, Lethal; D009154:Mutation; D010641:Phenotype; D020871:RNA Stability; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C111978:SKI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D063326:Exosome Multienzyme Ribonuclease Complex; C542283:DIS3 protein, S cerevisiae; C112610:RRP6 protein, S cerevisiae
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