The Yeast Exosome Functions as a Macromolecular Cage to Channel RNA Substrates for Degradation

Abstract

The exosome is a conserved macromolecular complex essential for RNA degradation. The nine-subunit core of the eukaryotic exosome shares a similar barrel-like architecture with prokaryotic complexes, but is catalytically inert. Here, we investigate how the Rrp44 nuclease functions in the active ten-subunit exosome. The 3.0 A resolution crystal structure of the yeast Rrp44-Rrp41-Rrp45 complex shows how the nuclease interacts with the exosome core and the relative accessibility of its endoribonuclease and exoribonuclease sites. Biochemical studies indicate that RNAs thread through the central channel of the core to reach the Rrp44 exoribonuclease site. This channeling mechanism involves evolutionary conserved residues. It allows the processive unwinding and degradation of RNA duplexes containing a sufficiently long single-stranded 3' extension, without the requirement for helicase activities. Although the catalytic function of the exosome core has been lost during evolution, the substrate recruitment and binding properties have been conserved from prokaryotes to eukaryotes.

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

“…The strong underrepresentation of C in the tails led us to assume that a high C-content might be unfavorable in this respect. Indeed, more than 90% of a GGG(CA) 15 transcript remained untouched by all three complexes ( Fig. 5A and B), although in similar experiments the 30-meric poly(A)-RNA was completely processed (Supplementary Fig.…”

“…For in vitro transcription of MCS-RNA 20 nucleotide (nt) and 30 nt variants, the primer 5 0 -TCGAGCAGCTGAAGCTTCCCTATAGTGAGTCGTATTA-3 0 and 5 0 -TCGA GCAGCTGAAGCTTGCATGCCTGCACCCTATAGTGAGTCGTATTA-3 0 were annealed with the T7-oligonucleotide (5 0 -TAATACGACTCACTA-TAGGG-3 0 ), and the MEGAshortscript™ Kit (Ambion) was used. Oligonucleotides with a 3 0 -sequence complementary to the T7-oligonucleotide were also used for the in vitro transcription of the GGG(CA) 15 and G(GU) 16 substrates. Heteropolymeric transcripts were internally labelled using [a-32 P] UTP or [a-32 P] ATP.…”

“…5. The vast majority of a GGG(CA) 15 -transcript is not processed by the exosome, while a G(GU) 16 -transcript is degraded or polyadenylated. Substrates (0.4 pmol per assay), processing products and protein complexes are indicated.…”

Poring over exosome structure

“…the crystal structures of overlapping parts of the eukaryotic exosome (Liu et al, 2006;Bonneau et al, 2009) and the related bacterial pNpase (Symmons et al, 2000) and archaeal exosome (Lorentzen et al, 2007) have been solved, and show that these rNa degrading machines from the three domains of life have a similar structure (Fig 1). they are all composed of a ring of six rNase pH domains, one side of which has a cap that contains putative rNa binding domains.…”

“…However, in humans and yeast each of the rNase pH domains have point mutations that make the exosome ring catalytically inactive (Dziembowski et al, 2007). instead, catalysis is carried out by a tenth subunit-rrp44/Dis3-which binds to the pH ring on the opposite side to the cap proteins (Bonneau et al, 2009;Wang et al, 2007). this organization made it unclear whether rNa also enters the central chan nel of the exosome in eukaryotes (Fig 1), or whether substrate rNas directly access the catalytic subunit.…”