Lee, Flora Chi Yan; (2019) Assembly of protein-RNA complexes relevant for neuronal function and ALS. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

RNA binding proteins (RBPs) play key regulatory roles throughout the life cycle of RNA molecules. In particular, post-transcriptional regulation by RBPs is pertinent to neuronal biology due to their extended morphology and consequently the need for local transcriptome and proteome remodelling at distal compartments. When bound to their RNA partners, collections of RBPs tend to assemble into higher-order ribonucleoprotein complexes (RNPs), but surprisingly little is known about the mechanisms that ensure that each mRNA molecule correctly assembles into a specific mRNP. RNP assembly is also a crucial puzzle in our understanding of neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS), where aberrant aggregation of RNPs is a hallmark of pathology and degeneration. Therefore I have focused on the study of the assembly of RNPs relevant for neuronal function and our understanding of ALS. The thesis has 3 parts, describing methods and insights pertaining to protein-RNA interactions, RNA-RNA interactions and the higher-order assembly of RNPs. First, I describe the optimisation of iCLIP (individual nucleotide-resolution crosslinking and immunoprecipitation), which identifies endogenous protein-RNA interactions in a transcriptome-wide manner. Second, I present hiCLIP (RNA hybrid iCLIP) studies of STAU2, a double-stranded RBP that controls localised mRNAs, identifying its physiological mRNA partners of STAU2 in rat brain. I comprehensively characterize the RNA duplexes that are bound by STAU2 in the cortex, which provides a view into the role of secondary structure of localised mRNAs in RNP assembly. Thirdly, using imaging methods, I assess the dynamics of nuclear TDP-43, an RBP with a central role in ALS, by using a panel of deletions and mutations in the C-terminal domain, to investigate how homomeric interactions affect RNP assembly. In summary, I have developed transcriptomic and imaging techniques to integrate our insights into the assembly of protein-RNA complexes, with implications for their neuronal function and role in neurodegenerative disease.

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