The Structural Analysis Of Yeast RNA Exosome With Cryo-EM

The eukaryotic RNA Exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein,which contains 3?-to-5? exo-and endo-RNase activities.Functional studies revealed Exosome as a critical player in the turn-over of almost all the RNA species including mRNAs,rRNA,tRNAs and other non-coding RNAs with the assistance from nuclear or cytoplasmic co-factors.Using single-particle EM and biochemical analysis,we provide visual evidence that two distinct substrate-recruitment pathways exist.In the through-core route,channeling of the single-stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44.In the alternative direct-access route,this conformational change does not take place,the RNA substrate is visualized to avoid the core and enter Rrp44's EXO site directly.Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic Exosome and indicate substrate-specific modes of degradation by this complex.Meanwhile,we used single particle cryo-electron microscopy(cryo-EM)obtained the structure of Exo10-Ski7 complex in its RNA-free and endogenous RNA-bound form at 4.2-Angstrom and 5.8-Angstrom resolution respectively.The high-resolution structures allowed us to depict the conformational difference between the two states in a near-atomic resolution level,providing reasonable insights on the mechanism of RNA-induced conformational change to activate the through-core RNA processing pathway.Furthermore,our structural analysis revealed the same Ski7 protein's N-terminal domain adopts a structural arrangement and interacts with Exosome's core complex in a similar fashion as Rrp6's C-terminal domain.This explains how distinct Exosome species in vivo are exclusively distinguished in different RNA processing events.