Structural and functional analysis of the ribosomal quality control trigger complex RQT

点击翻译按钮获取中文摘要

The translation of mRNA into proteins by ribosomes is a central process which is subject to errors and environmental impact. Thus, for numerous reasons this process can slow down or come to a halt resulting in stalled ribosomes that collide with trailing ones. These ribosomal collisions are recognized in order to be resolved and to serve as a proxy for problematic translation which elicits stress responses and triggers quality control pathways. A key step for collision clearance and a prerequisite to trigger ribosome associated quality control (RQC) is the dissociation of the stalled ribosomes into subunits which is facilitated by the ribosome associated quality control trigger complex RQT. This complex consists in yeast of three and in humans of up to four subunits (hRQT/ASC-1 complex) with a large Ski2-like helicase Slh1 (ASCC3 in humans). Slh1/ASCC3 contains two ATPase cassettes providing the central helicase activity required for ribosomal splitting. Upon ubiquitin modification of the small ribosomal subunit by the stall sensing E3 ligase Hel2 (ZNF598 in humans), RQT is recruited to the stalled ribosomes. Using biochemical assays and cryo-EM in the yeast system, we recently discovered that RQT binds to the small 40S ribosomal subunit and requires the emerging mRNA to remodel and thereby destabilize and split the collided ribosomes. Yet, the exact molecular mechanism of the splitting activity of RQT, e.g., its activation mechanism and the contribution of the individual potential helicase cassettes is not clear yet. Even less is known about the overall architecture of the human RQT complex (hRQT), its interaction with stalled ribosomes and its mode of function in collision dissociation. Therefore, we propose to investigate the molecular mechanism of RQT driven ribosomal splitting in the yeast and the human system. Using mainly in vitro reconstitution approaches and assays, we will analyze the RNA helicase activity of the individual N- and C-terminal ATPase cassettes of Slh1. In addition, we will use cryo-EM in order to directly visualize mRNA-bound RQT on the yeast ribosome by employing different mRNA trapping conditions. In the human system we will directly visualize by cryo-EM the trimeric and tetrameric hRQT in order to gain insights in its overall architecture and potential autoregulatory features. Moreover, we suggest to use in vitro reconstitution assays and in vivo pullouts under hRQT-enriching conditions to visualize hRQT bound to collided human ribosomes, representing distinct intermediate states of the splitting process. Together, this will provide us with biochemical and structural information required to understand the molecular mechanisms employed by RQT/hRQT for the clearance of collided ribosomes and trigger of quality control pathways. The results may also be of value to better understand the underlying mechanisms of pathologies such as certain types of cancer that are caused by a dysfunctional hRQT system.