B01: Clearance of defective mRNA by-products by the ribosome-associated quality control pathway in stress and disease

The ribosome represents as a central hub for mRNA and protein quality control. Translation of defective mRNA species, such as damaged or truncated transcripts, often results in abnormally long pauses in ribosome elongation that trigger mRNA silencing/decay and nascent protein degradation. The ribosome-associated protein quality control (RQC) pathway marks elongation-stalled nascent amino acid chains for degradation while they are still associated with the ribosome, thereby limiting their toxic effects. RQC disruption sensitizes cells to translational stress and, at the organismal level, promotes early onset motor- and neurodegenerative phenotypes. Although the mechanistic aspects of the RQC pathway have been characterized in detail, little is known about the physiological triggers of RQC that permeate these phenotypes, or what are the specific consequences of RQC disruption to the fidelity of the proteome. In the present project, we will systematically characterize defective mRNA species arising upon proteotoxic stress at the endoplasmic reticulum (ER), as well as the impact of RQC on the maintenance of proteome fidelity during ER stress. We postulate that ER stress might not only exacerbate common mRNA processing mistakes such as premature polyadenylation, but also induce the formation of truncated mRNAs via endonucleolytic cleavage. Cells respond to proteotoxic ER conditions by activating stress response pathways that mediate the degradation of ER-localized mRNAs to acutely decrease secretory/membrane protein synthesis, thereby reducing the burden on the proteostasis network. Both RIDD (regulated IRE1-dependent decay) and ERAS (ER-associated RNA silencing) pathways promote the degradation of transcripts translated at the ER surface; whether their activity interferes with ribosomal elongation remains to be tested. Therefore, we will identify mRNA targets of RIDD and ERAS in HEK293 human cells exposed to ER stress and test if their decay is coupled to co-translational nascent chain degradation by the RQC or related pathways. Moreover, we will use LiP-MS to compare the conformational status of the proteome in wild type and RQC knockout cells in conditions of ER stress. By integrating the results of transcriptomic and proteomic experiments we envision providing a mechanistic understanding of the physiological causes and consequences of RQC, thereby clarifying its role in the maintenance of proteome homeostasis upon loss of mRNA fidelity.