[F31] Deciphering RNA Zipcodes: A Novel Framework for Subcellular Localization Element Annotation

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Abstract: The spatial organization of mRNAs within cells is a critical yet underexplored layer of gene regulation. Across diverse biological contexts, localized RNA translation enables rapid and spatially-precise protein production. This function is crucial for enabling differentiation in embryonic morphogenesis, nutrient uptake in the intestinal epithelium, and the dynamic modulation of synaptic activity and plasticity in neurons. Despite its importance, the mechanistic study of RNA localization has historically been limited by a lack of scalable tools. Imaging studies first illustrated that transcripts accumulate in distinct subcellular regions, but broad profiling only became feasible with the advent of proximity labeling and sequencing approaches capable of mapping thousands of mRNAs at once. A key unanswered question is how each transcript “knows” where to go. One prevalent mechanism involves sequences within the transcript called “zipcodes”, typically found within the 3′ untranslated region, which bind RNA-binding proteins (RBPs) to guide transcripts along the cytoskeleton to subcellular compartments. Our lab’s data have revealed over a thousands of transcripts within cells localize to distinct locales, including the outer mitochondrial membrane (OMM). Notably, several nuclear-encoded RNAs require accurate mitochondrial targeting for proper oxidative phosphorylation, autophagy, and apoptosis – critical processes often dysregulated in cancer and neurodegenerative diseases. To identify zipcodes on a large scale and elucidate their contribution to cellular function and disease, I will integrate massively parallel reporter assays and APEX-seq, enabling both large-scale annotation of unknown zipcodes and high-resolution localization profiling. I will also employ a BirA-BoxB proximity labeling strategy to pull down and identify RBPs that interact directly with these zipcodes. These combined approaches will reveal the cis- and trans-acting factors that facilitate mRNA transport. Finally, I will test the pathogenic potential of dysfunctional zipcodes. I will interrogate clinically relevant single-nucleotide polymorphisms (SNPs) in nuclear-encoded mitochondrial genes to determine whether these variants cause mislocalization of mitochondrial transcripts. SNP introduction and mislocalization will be validated with CRISPR prime editing and fluorescent in situ hybridization imaging, respectively. Functional assays – such as for investigating oxygen consumption rate, ATP production, and ROS levels – will clarify how mislocalization impairs normal protein function. Collectively, this work will deliver a comprehensive catalog of zipcodes directing mitochondrial mRNA localization, reveal their RBP partners, and elucidate how mutations affecting mRNA localization can drive disease. Our research ultimately represents a high-impact, easily reproducible, and innovative framework for transcriptome-wide zipcode element annotation in living cells which may be adap Istituzione: BAYLOR COLLEGE OF MEDICINE PI: Steven Nguyen Progetto: 1F31CA314447-01

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Source: reporter.nih.gov