Deadenylation-dependent RNA decay in eukaryotes

RNA abundance is regulated by balancing transcription and degradation, processes that control the temporal and spatial distribution of cellular RNA. In eukaryotic cells, mRNA decay is catalyzed by two major pathways, and both can be initiated by deadenylation of the polyadenylated (poly-A) tail. After decapping, 5’ to 3’ RNA degradation is accomplished by Xrn1, a 5’ to 3’ exoribonuclease. In the 3’ to 5’ pathway, RNA degradation is catalyzed by a multi-subunit 3’ to 5’ exoribonuclease complex, the RNA exosome. The exosome also participates in processing small nucleolar and small nuclear RNAs (snoRNAs, snRNAs) and ribosomal RNAs (rRNAs). Exosome activities play a role in nonsense mediated decay, non-stop decay, and no-go decay, and are regulated in part through association with complexes such as Ski7p and the Ski2p/Ski3p/Ski8p complex and TRAMP, a multi-subunit polymerase complex that primes structured RNA for degradation.

Composition of the eukaryotic exosome

The eukaryotic RNA exosome is composed of nine to eleven distinct gene products, several of which share sequence similarity to bacterial 3’ to 5’ exoribonucleases. Six exosome subunits, Rrp41, Rrp42, Rrp43 (OIP2 human), Rrp45 (PM/Scl-75 human), Rrp46, and Mtr3, share as much as 20-30% sequence identity to E. coli RNase PH and PNPase, two processive, phosphorolytic 3’ to 5’ exoribonucleases that activate inorganic phosphate as a nucleophile to release ribonucleoside 5’ diphosphate products. Three additional exosome subunits, Csl4, Rrp4, and Rrp40, include S1 or KH domains which are postulated to bind RNA. The tenth yeast exosome subunit is Rrp44, a processive hydrolytic exoribonuclease that shares sequence similarity to E. coli RNase R and RNase II. Rrp44 appears integral to the yeast exosome, although human Rrp44 (Dis3) does not appear as a stochiometric component in human exosome preparations, suggesting that the composition of exosomes in human and yeast may differ. The tenth human subunit and eleventh yeast subunit is Rrp6 (PM/Scl-100 human), a distributive hydrolytic exoribonuclease that shares sequence similarity to E. coli RNase D. Rrp6 is not essential, is associated with nuclear exosomes in yeast and human, although Rrp6 may not be restricted to the nucleus.

Structure of the human 9-subunit exosome

Structures of RNA exosomes

We determined the activities and structure of the eukaryotic exosome by reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. No activities were observed for the 9-subunit yeast exosome, although processive hydrolytic activities were observed for yeast Rrp44 and the yeast 10-subunit exosome, and distributive hydrolytic activities were observed for Rrp6. Both processive and distributive hydrolytic activities were observed for the yeast 11-subunit exosome. We determined the overall architecture of the eukaryotic exosome by determining the X-ray structure for the nine-subunit human exosome.

Our investigation of the exosome core in budding yeast revealed that its central channel is essential, and that it functions to guide RNA substrates to the active sites of both Rrp44 and Rrp6, albeit with differing dependencies. We subsequently determined the structure of the yeast exosome core in complex with Rrp6 and polyA RNA. This work illuminated RNA contacts to the exosome core that influenced Rrp6 activities in both yeast and human systems.