Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex

Abstract

The exosome is the major 3'-5' RNA-degradation complex in eukaryotes. The ubiquitous core of the yeast exosome (Exo-10) is formed by nine catalytically inert subunits (Exo-9) and a single active RNase, Rrp44. In the nucleus, the Exo-10 core recruits another nuclease, Rrp6. Here we crystallized an approximately 440-kilodalton complex of Saccharomyces cerevisiae Exo-10 bound to a carboxy-terminal region of Rrp6 and to an RNA duplex with a 3'-overhang of 31 ribonucleotides. The 2.8 Å resolution structure shows how RNA is funnelled into the Exo-9 channel in a single-stranded conformation by an unwinding pore. Rrp44 adopts a closed conformation and captures the RNA 3'-end that exits from the side of Exo-9. Exo-9 subunits bind RNA with sequence-unspecific interactions reminiscent of archaeal exosomes. The substrate binding and channelling mechanisms of 3'-5' RNA degradation complexes are conserved in all kingdoms of life.

CryoEM structure of yeast cytoplasmic exosome complex

“…As we and others have previously discovered, RNA molecules with a long enough single-strand (ss) 3 overhang induce a major rearrangement of the Rrp44 RNase II-like domain relative to the core complex so that the ssRNA substrates coming out of the core directly thread into the exonuclease active site for degradation [13][14][15]17]. However, how this major conformational change in Rrp44 occurs upon RNA substrate binding was unknown.…”

“…Similar to its prokaryotic and archaeal homologs, the eukaryotic Exo10 can recruit RNA substrates with single-stranded 3′ overhangs via the RNA-binding proteins at the top of its core and move the substrates entirely through the central channel and to the RNase active site of Rrp44 below the core. This through-core pathway, supported by the crystal structure of the holo-enzyme bound to an RNA substrate [14], requires the RNA substrate to have a single-stranded 3′ overhang longer than 30 nt to reach the Rrp44 exonuclease active site from the entry point of the core. The solvent exposure of the Rrp44 active site in the apo structure of Exo10 also suggests another pathway where RNA substrates reach the Rrp44 active site without passing through the central channel of the core.…”

“…To determine which site is more critical for the conformational change, we designed two hairpin RNAs with single-stranded 3′ overhangs of 24 nt (RNA24) or 18 nt (RNA18), and examined their abilities to induce exosome conformational change. According to the crystal structure of the holo-Exo10-Rrp6-RNA complex [14], RNA24 should be able to reach the bottom region of the central channel at Site 1 but should not reach Site 2, whereas the tail of RNA18 should be too short to even reach Site 1. We incubated these substrates with the Exo10 complex (in a preparation free of any endogenously bound RNAs, generated by the aforementioned MgCl 2 treatment procedure) and performed single-particle EM analysis of the final complex.…”

“…Rrp44 is a multi-domain protein with a processive hydrolytic exonuclease activity at its C-terminal region, which is homologous to bacterial RNase II, and a relatively weak endonuclease activity at its N-terminal region [10][11][12]. Previous structural studies have shown that Rrp44 specifically interacts with Rrp41, Rrp43 and Rrp45 of the core complex, aligning with the central channel's exit of the core complex [13][14][15]. The Rrp44-bound exosome complex is considered to be an important active core complex in eukaryotes and is termed Exo10 [16].…”

“…In the best studied Saccharomyces cerevisiae system, Rrp6, an RNase D-type enzyme with distributive exonuclease activity, interacts physically and functionally with the exosome in the nucleus [18]. Rrp6 has been revealed to interact with the Exo10 complex at the top region, next to the entry site of the core [14,15,19]. In the cytoplasm, Ski7, a putative GTPase, bridges the exosome with the ribosome complex and the Ski2/3/8 complex, a cofactor complex, thus promoting recognition and degradation of aberrant mRNAs by the exosome [20].…”