RNA localization is an evolutionarily conserved mechanism for the spatiotemporal regulation of protein synthesis. Secretory and membrane protein-encoding mRNAs are the largest cohort of localized mRNAs, comprising approximately 35% of protein-encoding genes, and are thought to undergo co-translational localization to the endoplasmic reticulum (ER) via the signal recognition particle (SRP) pathway. While SRP pathway function in protein translocation is well accepted, a companion role in mRNA localization on the ER has not been established. Strikingly, recent studies revealed that all mRNAs can be locally translated on the ER, indicating an unexpected diversity in ER-localized gene expression and suggesting that multiple, though as yet unknown, pathways function in RNA localization to the ER. To this end, work from our lab and others have provided new evidence that RNA binding proteins (RBPs) contribute key functions in mRNA localization to the ER. However, our understanding of how RBPs mediate mRNA transport to the ER for regulated translation is very limited. Here, we propose to uncover the molecular mechanisms of ER-specific mRNA regulation by RBPs, using leucine-rich repeat containing 59 (LRRC59) as a model. LRRC59 is a highly conserved and constitutively expressed ER-resident protein known to bind the ribosome and RNA. Using proximity proteomics, we recently discovered that LRRC59 also interacts with SRP factors and translation machinery. From these data, we hypothesize that LRRC59 functions in compartmentalized mRNA regulation by anchoring target mRNAs and ribosomes in close proximity to facilitate efficient protein synthesis. To test this hypothesis, we will combine advanced deep sequencing approaches with optimized subcellular fractionation methods on wild type and LRRC59 knockout models to: (1) identify the LRRC59- associated RNA transcriptome, and (2) investigate the molecular mechanism of LRRC59-regulated translation on the ER. Given the importance of ER stress and translational re-programming on human health, we further hypothesize that LRRC59 may have a role in maintaining cellular proteostasis. Using transcriptome-wide sequencing approaches and optical imaging technologies, we will be able to address how ER translation is regulated during ER stress, both globally and in a LRRC59-specific manner. Collectively, we expect this work to reveal new mechanisms that govern compartmentalized mRNA localization and translation in normal human physiology and disease. Importantly, the proposed studies will enhance my training in multiple aspects of RNA biology and RNA-related technologies, and provide me with the necessary foundation to build an independent research program.
Mounting evidence suggests a role for RNA binding proteins (RBPs) in mRNA regulation on the endoplasmic reticulum (ER); however, the molecular mechanisms of these processes remain largely uncharacterized. Here, we aim to identify new paradigms for RBP-mediated subcellular mRNA localization and translation, using an ER-resident RBP, LRRC59 as a model. Using a multidisciplinary experimental approach, this research is expected to uncover novel mechanisms of post-transcriptional gene regulation, characterize the ER-specific transcriptome and translatome, and significantly advance understanding of how RNA dysregulation on the ER contributes to human disease.
