Heterogeneous complexes of the RNA exosome in Sulfolobus solfataricus.

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.

Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes.

The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e. chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes.

The archaeal exosome localizes to the membrane.

We studied the cellular localization of the archaeal exosome, an RNA-processing protein complex containing orthologs of the eukaryotic proteins Rrp41, Rrp42, Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. Fractionation of cell-free extracts of Sulfolobus solfataricus in sucrose density gradients revealed that DnaG and the active-site comprising subunit Rrp41 are enriched together with surface layer proteins in a yellow colored ring, implicating that the exosome is membrane-bound. In accordance with this assumption, DnaG and Rrp41 were detected at the periphery of the cell by immunofluorescence microscopy. Our finding suggests that RNA processing in Archaea is spatially organized.

The evolutionarily conserved subunits Rrp4 and Csl4 confer different substrate specificities to the archaeal exosome.

We studied the substrate specificity of the exosome of Sulfolobus solfataricus using the catalytically active Rrp41-Rrp42-hexamer and complexes containing the RNA-binding subunits Rrp4 or Csl4. The conservation of both Rrp4 and Csl4 in archaeal and eukaryotic exosomes suggests specific functions for each of them. We found that they confer different specificities to the exosome: RNA with an A-poor 3'-end is degraded with higher efficiency by the Csl4-exosome, while the Rrp4-exosome strongly prefers poly(A)-RNA. High C-content and polyuridylation negatively influence RNA processing by all complexes, and, in contrast to the hexamer, the Rrp4-exosome prefers longer substrates.

Rrp4 and Csl4 are needed for efficient degradation but not for polyadenylation of synthetic and natural RNA by the archaeal exosome.

The exosome of the archaeon Sulfolobus solfataricus is a protein complex with phosphorolytic and polyadenylating activity. Little is known about its substrates and the regulation of its functions. We characterized the catalytically active hexameric ring composed of SsoRrp41 and SsoRrp42, and the nine-subunit exosomes containing in addition RNA binding protein SsoRrp4 or SsoCsl4 under various reaction conditions. The exosome synthesized heteropolymeric RNA tails and exhibited the highest in vitro activity at 60-70 degrees C. MgCl(2) was necessary for exosome activity. The two reactions, degradation and polyadenylation of RNA, were inhibited by increasing glycerol and KCl concentrations but were differently influenced by changes in pH and by increasing MgCl(2) concentrations. The three protein complexes with different compositions were similarly influenced by increasing concentrations of glycerol, KCl, and MgCl(2), but the SsoRrp4 exosome behaved differently with respect to pH changes. A 20-nucleotide poly(A) tail enabled the degradation and the polyadenylation of a 16S rRNA-derived transcript by the hexamer. Generally, RNA synthesis by the hexamer was more efficient than RNA phosphorolysis. Single-stranded poly(A) RNA, a heteropolymeric 97-nucleotide transcript, and natural tRNA were quickly polyadenylated, showing that these substrates were bound and their 3'-ends reached the active site. Despite this, their efficient degradation was possible only in the presence of SsoRrp4 or SsoCsl4. Thus, strong substrate binding by SsoRrp4- or SsoCsl4-containing exosomes is more important for phosphorolysis than for tailing of RNA. In summary, the data suggest that subunit composition and Mg(2+) are involved in the regulation of exosome activity.