Regulation of ribosome collisions during health, development and disease - PROJECT SUMMARY/ABSTRACT Obstacles on mRNAs cause eukaryotic ribosomes to stall. Severely stalled ribosomes collide with the upstream, translating ribosomes and form a complex of two ribosomes, referred to as “disome”, which is detected and removed by the Ribosome Quality Control (RQC) pathway. By establishing Disome-seq technique, which reveals genome-wide distribution of ribosome collisions across the transcriptome, we showed that RQC-targeted disomes frequently form on yeast mRNAs. We also found that collisions in yeast trigger the Integrated Stress Response (ISR), which reduces translation while inducing the expression of survival genes. Similarly, disomes activate ISR in humans to promote survival, but its hyperactivation leads to cell death. This indicates that cellular health is determined by disome homeostasis, which collectively refers to the disome distribution across transcripts, the overall disome abundance and the downstream response evoked by RQC/ISR. The mechanisms by which RQC and ISR detect disomes and affect collision dynamics, however, are poorly understood. The mutations of RQC and ISR components as well as dysregulated ribosome stalling have been linked to neurodevelopmental and neurodegenerative diseases. However, the contribution of disome homeostasis to neuronal differentiation and function is unclear. The objective of this proposal is to determine the mechanisms of disome homeostasis, to examine the role of disome regulation in neurons and to understand how its dysregulation leads to diseases. I hypothesize that regulation of disome homeostasis is important for neurodevelopment and neuronal health, and its dysregulation causes neuronal pathologies. In Aim 1, I will determine the mechanism of disome recognition by RQC and ISR in healthy and stressed cells by using selective Disome-seq and single-molecule fluorescence microscopy. In Aim 2, I will determine the distribution and abundance of disomes, and the role of RQC/ISR factors during the differentiation of induced pluripotent stem cells (iPSCs) into mature neurons by using fluorescence reporters, Disome-seq and neuronal assays. In Aim 3, I will delineate the link between dysregulation of disome homeostasis and neuronal diseases that were proposed to affect ribosome stalling by using Disome-seq, RNA-seq, RQC/ISR reporters and phenotypic experiments in the cells with disease mutations. I will further test the translation inhibitors as potential therapeutics to ameliorate the disease phenotypes. Overall, proposed work will illuminate crucial insights into the regulation of ribosome collisions and their role in neuronal homeostasis.
