Structural basis for clearing of ribosome collisions by the RQT complex

Abstract

Translation of aberrant messenger RNAs can cause stalling of ribosomes resulting in ribosomal collisions. Collided ribosomes are specifically recognized to initiate stress responses and quality control pathways. Ribosome-associated quality control facilitates the degradation of incomplete translation products and requires dissociation of the stalled ribosomes. A central event is therefore the splitting of collided ribosomes by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryogenic electron microscopy of RQT-ribosome complexes reveals that RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose that the Ski2-like helicase 1 (Slh1) subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the small ribosomal subunit, ultimately resulting in subunit dissociation. Our findings provide conceptual framework for a helicase-driven ribosomal splitting mechanism.

Extended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex

“…Recent cryoEM structures of yeast RQT–ribosome complexes revealed that prior to ribosome splitting the yeast ASCC3 ortholog, Slh1p, can adopt a more open conformation with fewer direct interactions between the two helicase cassettes as observed in our human ASCC3–TRIP4 complex structure 25 . While in the imaged conformations both Slh1p helicase cassettes are potentially accessible to an RNA substrate, no corresponding substrate density was observed at either Slh1p cassette 25 .…”

“…Recent cryoEM structures of yeast RQT–ribosome complexes revealed that prior to ribosome splitting the yeast ASCC3 ortholog, Slh1p, can adopt a more open conformation with fewer direct interactions between the two helicase cassettes as observed in our human ASCC3–TRIP4 complex structure 25 . While in the imaged conformations both Slh1p helicase cassettes are potentially accessible to an RNA substrate, no corresponding substrate density was observed at either Slh1p cassette 25 . In the observed conformations, mRNA could apparently be accommodated directly at the Slh1p CC, but an Slh1p variant harboring an ATPase-deficient NC (Slh1p K361R ) was required to capture RQT–ribosome complexes at a stage preceding ribosome splitting 25 , indicating that the NC ATPase/helicase activity is also required for the splitting reaction.…”

“…E.g., reduced ribosome association was found for an ASCC3 variant with an ATPase-deficient NC 23 , and selective abrogation of the ATPase activity in the ASCC3 NC or CC led to reduced disruption of stalled ribosomes 24 . Likewise, the ATPase activity of the Slh1p NC was shown to be required for ribosome dissociation 26 , 27 , 41 or completion of subunit splitting 25 .…”

“…The RQT complex aids in resolving stalled di-ribosomes or polysomes arising during aberrant translation, where it ultimately splits the stalled lead ribosomes into subunits 23 , 24 . An analogous RQT complex, comprising RQT2 (ASCC2 ortholog), Slh1p/RQT2 (ASCC3 ortholog), and RQT4 (TRIP4 ortholog) has been identified in yeast 25 – 28 .…”

“…Moreover, while ASCC3 has originally been described as a DNA helicase, it is thought to translocate on mRNA or possibly rRNA during ribosome quality control. However, in a recent structural analysis of yeast RQT–ribosome complexes, RNA binding to either Slh1p helicase cassette remained unresolved 25 . Thus, it is presently unclear whether during ASCC3-related processes the cassettes engage continuous or discontinuous regions of a nucleic acid substrate or perhaps even different nucleic acid molecules, and how their helicase activities may be coordinated.…”