The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.
The eukaryotic RNA exosome is an essential and conserved protein complex that can degrade or process RNA substrates in the 3 ′ -to-5 ′ direction. Since its discovery nearly two decades ago, studies have focused on determining how the exosome, along with associated cofactors, achieves the demanding task of targeting particular RNAs for degradation and/or processing in both the nucleus and cytoplasm. In this review, we highlight recent advances that have illuminated roles for the RNA exosome and its cofactors in specific biological pathways, alongside studies that attempted to dissect these activities through structural and biochemical characterization of nuclear and cytoplasmic RNA exosome complexes.
SUMMARY
The eukaryotic RNA exosome is an essential and conserved 3’ to 5’ exoribonuclease complex that degrades or processes nearly every class of cellular RNA. The nuclear RNA exosome includes a nine-subunit non-catalytic core that binds Rrp44 (Dis3) and Rrp6 subunits to modulate their processive and distributive 3’ to 5’ exoribonuclease activities, respectively. Here, we utilize an engineered RNA with two 3’ ends to obtain a crystal structure of an eleven-subunit nuclear exosome bound to RNA at 3.1 Å. The structure reveals an extended RNA path to Rrp6 that penetrates into the non-catalytic core, contacts between the non-catalytic core and Rrp44 that inhibit exoribonuclease activity, and features of the Rrp44 exoribonuclease site that support its ability to degrade 3’ phosphate RNA substrates. Using reconstituted exosome complexes, we show that 3’ phosphate RNA is not a substrate for Rrp6, but is readily degraded by Rrp44 in the nuclear exosome.
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