Cellular response to small molecules that selectively stall protein synthesis by the ribosome.

Identifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease. Author summary A fundamentally new approach to treat human diseases caused by undruggable proteins would be to use small molecules to selectively inhibit their synthesis by the ribosome. Here we compare two related compounds (PF846, PF8503) that selectively stall human translation to the effects of a more general translation inhibitor, homoharringtonin. We used genome-wide approaches to probe the effects of these compounds, including measurements of which messenger RNAs are being translated and the effects of knocking down gene expression on cell growth. These experiments revealed new and surprising genetic connections between ribosome quality control pathways. We then used biochemical and cell-based experiments to test the involvement of particular ribosome quality control proteins such as ASCC2, ASCC3 and HBS1L in the physiological response of cells to translation inhibitors. The genetic and biochemical insights presented here should aid the development of next-generation selective translation inhibitors to treat disease.