Understanding genome-wide and single-molecule dynamics of colliding ribosomes during health and disease

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PROJECT SUMMARY/ABSTRACT Eukaryotic ribosomes translating defective mRNAs, such as those that are damaged, “stall” and become unavailable for new rounds of translation. The ribosome-associated quality control (RQC) system detects the stalled complex formed by two collided ribosomes (“disome”) and degrades the defective mRNAs to prevent aberrant translation. Since this pathway leads to irreversible mRNA decay, it is critical for the RQC machinery to differentiate functional ribosome pausing events, from aberrant ribosome stalling cases that need to be resolved. However, previous RQC studies used artificial mRNA substrates that induce extreme cases of ribosome stalling. Therefore, it is unknown how frequently and where disomes form on regular transcripts. It is also unclear if RQC recognizes all disomes and what the cellular consequences of recognition are. Interestingly, impaired ribosome collisions have been linked to a neurodevelopmental disorder, Fragile-X syndrome (FXS), which is the most common form of inherited intellectual disability. Nevertheless, the pathological dynamics of ribosome collisions during FXS as well as the interplay between collisions and RQC pathway during neurodevelopment have been poorly studied. There is a critical need, therefore, to determine the dynamics of ribosome collisions in healthy cells and to understand how their dysregulation leads to FXS. The objective of this proposal is to determine the role of ribosome collisions during neurodevelopment. My hypothesis is that under physiological conditions, RQC-targeted disomes form on regular transcripts and they maintain neuronal homeostasis by regulating protein expression. I further postulate that dysregulation of disome formation leads to cellular stress and causes disease phenotypes. To test this idea, in aim 1, I will determine the genome-wide distribution of disomes in human cells using the Disome-seq technique that we recently established in our lab. To visualize the dynamics of RQC upon ribosome collision, in aim 2, I will monitor real-time regulation of RQC using cutting-edge dual-color single molecule imaging in human cells. To understand the functional role of ribosome collisions, in aim 3, I will characterize the link between dysregulated ribosome stalling in an FXS model of neuronal differentiation. I will further study the action of translation inhibitors for their potential of restoring the collisions in neurons. Overall, the proposed studies aim to determine the prevalence of ribosome collisions in the cell and how dysregulation of these collisions can cause neurodevelopmental defects.