title: Heterogeneous complexes of the RNA exosome in Sulfolobus solfataricus.
abstract: The archaeal exosome is a protein complex involved in the degradation and the posttranscriptional tailing of RNA. The proteins Rrp41, Rrp42, Rrp4, Csl4 and DnaG are major subunits of the exosome in Sulfolobus solfataricus. In vitro, Rrp41 and Rrp42 form a catalytically active hexamer, to which an RNA-binding cap of Rrp4 and/or Csl4 is attached. Rrp4 confers strong poly(A) specificity to the exosome. The majority of Rrp41 and DnaG is detectable in the insoluble fraction and is localized at the cell periphery. The aim of this study was to analyze whether there are differences in the composition of the soluble and the insoluble exosomes. We found that the soluble exosome contains less DnaG and less Csl4 than the insoluble exosome which co-sediments with ribosomal subunits in sucrose density gradients. EF1-alpha was co-precipitated with the soluble exosome from S100 fractions using DnaG-directed antibodies, and from density gradient fractions using Rrp41-specific antibodies, strongly suggesting that EF1-alpha is an interaction partner of the soluble exosome. Furthermore, Csl4 was co-immunoprecipitated with the exosome using Rrp4-specific antibodies and vice versa, demonstrating the presence of heteromeric RNA-binding caps in vivo. To address the mechanism for poly(A) recognition by Rrp4, an exosome with an RNA-binding cap composed of truncated Rrp4 lacking the KH domain was reconstituted and analyzed. Although the deletion of the KH domain negatively influenced the degradation activity of the exosome, the poly(A) specificity was retained, showing that the KH domain is dispensable for the strong poly(A) preference of Rrp4.
authors.affiliation: Institute of Microbiology and Molecular Biology, Heinrich-Buff-Ring 26-32, D-35392 Gießen, Germany.
authors.forename: Chamindri
authors.initials: C
authors.lastname: Witharana
authors.forename: Verena
authors.initials: V
authors.lastname: Roppelt
authors.forename: Günther
authors.initials: G
authors.lastname: Lochnit
authors.forename: Gabriele
authors.initials: G
authors.lastname: Klug
authors.forename: Elena
authors.initials: E
authors.lastname: Evguenieva-Hackenberg
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D011061:Poly A