Insights into the Mechanism of Progressive RNA Degradation by the Archaeal Exosome

Abstract

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.Initially described as a multisubunit RNase complex required for maturation of 5.8 S rRNA in yeast (1), the 3Ј 3 5Ј exoribonuclease complex exosome was subsequently shown to play a central role on numerous pathways related to RNA processing and degradation, both in the nucleus and in the cytoplasm (reviewed in Ref. 2). These include 3Ј end processing of rRNAs, small nuclear RNAs, and small nucleolar RNAs (3-5), degradation of aberrant pre-mRNAs, pre-tRNAs, and pre-rRNAs (6 -9), normal turnover of cytosolic mRNAs (10), and degradation of RNA fragments produced during RNA interference processes (11, 12).Increasing structural and genetic studies with archaeal and eukaryotic exosomes have unveiled the evolutionary conservation of its molecular architecture (1, 13-18). The archaeal exosome consists of two RNase PH subunits (Rrp41 and Rrp42) and two proteins containing the S1/KH (Rrp4) or S1/zinc ribbon (Csl4) RNA-binding domains (17)(18)(19). Determination of the three-dimensional structure of the Sulfolobus solfataricus and Archaeoglobus fulgidus exosomes revealed a PNPase-like fold (20), composed by alternating RNase PH subunits assembled into a hexameric ring capped by a trimer of RNA-binding proteins, which can be formed by either Rrp4 or Csl4 or possibly by a mixture of both (19,21,22). Such an architecture encloses the exoribonucleolytic active sites at the bottom of the RNase PH ring catalytic chamber and restricts the entry to only unstructured RNA through the S1 pore, formed by the RNAbinding subunits of the exosome cap placed at the top of the ring (19,(21)(22)(23). Both Rrp41 and Rrp42 subunits possess the same RNase PH-fold and are involved in substrate binding, but the amino acid residues contributing to the interactions with the phosphate nucle...

Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation

“…A similar structural organization is observed in the archaeal exosome core complex, in which the two archaeal RNase PH family proteins, Rrp41 and Rrp42, form a hexameric ring composed of three copies of Rrp41-Rrp42 heterodimers (Buttner et al 2005;Navarro et al 2008). The phosphorolytic exonuclease active sites of the archaeal exosome are situated in the internal ''processing chamber'' of the Rrp41-Rrp42 ring.…”

“…The phosphorolytic exonuclease active sites of the archaeal exosome are situated in the internal ''processing chamber'' of the Rrp41-Rrp42 ring. Only Rrp41 has an RNase active site, whereas Rrp42 is required for complex assembly and activity (Buttner et al 2005;Navarro et al 2008). On the other hand, the recombinant yeast and human exosome cores are inactive and lack any detectable phosphorolytic exoribonuclease activity, suggesting that all of the RNase PH proteins in the ring, including hRrp46, are inactive .…”

Crystal structure of Escherichia coli PNPase: Central channel residues are involved in processive RNA degradation

“…Previously published crystal structures of an archaeal exosome in complex with RNA substrates show a ribonucleotide bound at the narrowest constriction of the central channel (Lorentzen et al 2007;Navarro et al 2008). To find out if RNA is also bound at the neck regions in PNPase, we mutated the two arginine residues located at the upper neck (R102 and R103) closer to the channel entrance, and one arginine located at the lower neck (R106) closer to the active site, to generate the double-mutant R102A/R103A and a single-point mutant R106A.…”

“…The crystal structures of archaeal exosome in complex with tungstate (mimicking phosphate), and in complex with RNA, further suggest that the single-stranded RNA substrate is threaded through the central channel of the ring-like structure, as tungstates and 4-nucleotide (nt) RNAs are bound at the three active sites buried inside the channel (Buttner et al 2005;Navarro et al 2008). The threading model provides the basis for the discrimination between structured and unstructured RNAs by PNPase and exosomes since they only degrade single-stranded RNAs.…”

“…The threading model provides the basis for the discrimination between structured and unstructured RNAs by PNPase and exosomes since they only degrade single-stranded RNAs. Moreover, the crystal structure of the archaeal exosome bound with RNA substrates shows fragment RNA bound both at the active site and at the narrowest constriction of the central channel near the channel entrance (Lorentzen et al 2007;Navarro et al 2008). These results demonstrate that singlestranded RNAs are indeed threaded through the channel, particularly bound at the constricted neck region.…”