Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus

Abstract

SummaryThe eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.

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

“…2 and Ref. [9]). We conclude that S. solfataricus Csl4 is helpful for the interaction of A-poor RNAs, while Rrp4 confers poly(A)-specificity to the exosome.…”

“…Labelling of 30-meric poly(A) [9] at the 5 0 -end, in vitro transcription of internally labelled MCS-RNA derived from the multiple cloning site of pSP72 and of other short, heteropolymeric transcripts, and purification of substrates through denaturing gels were previously described [10]. To synthesize tailed MCS-RNA variants, T7-promotor containing PCR products were generated with T7-forward-primer (5 0 -TAATACGACTCACTATAGGG-3 0 ) and one of the following oligonucleotides (MWG Operon) 5 0 -(T) 20 CGAGCAGCTGAA GCTT-3 0 for MCS-RNA(A) 20 , 5 0 -(A) 20 TCGAGCA-GCTGAAGCTT-3 0 for MCS-RNA(U) 20 , and 5 0 -CTCCTTTTATAATTATCCCTCGAGCAGCT-GAAGCTTG-3 0 for MCS-RNA 19hetero .…”

Global Analysis of mRNA Decay in Halobacterium salinarum NRC-1 at Single-Gene Resolution Using DNA Microarrays

“…As the degradosome and the eukaryotic exosome, it includes RNase PH domain proteins. Besides the exoribonucleolytic activity the archaeal exosome also exhibits polyadenylation activity (27,34,48). Remarkably, genes encoding the counterparts of the core subunits of this exosome are not present in the genomes of Halobacterium salinarum NRC-1 and Methanocaldococcus jannaschii (22).…”

Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis

“…The DnaG-like primase was reported to play a role in RNA degradation rather than DNA replication (5,6), and genetic studies suggest that the genes encoding each of the eukaryotic-like primase subunits are essential, whereas the dnaG gene is dispensable for cell growth (7). The archaeal primase contains a two-subunit structure with significant homology to the eukaryotic p58 and p48 subunits.…”