Capitanchik, Charlotte; (2022) Computational studies of RNA modification-dependent RNA binding protein networks. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The covalent modification of RNA nucleotides is a powerful layer of post-transcriptional control of gene expression across the tree of life. Historically, only abundant modifications on abundant RNAs such as tRNA and rRNA could be studied, due to methodological limitations. In the past decade, leaps forward in biochemistry and high throughput sequencing methods have enabled mapping of RNA modifications across all RNA species. In particular this thesis focuses on the most abundant internal modification of mRNA, N6-methyladenosine (m6A), and how RNA binding proteins (RBPs) interact with RNA modifications to impact RNA life cycle. Alongside these experimental developments have come new computational challenges. Integration of many datasets must be approached carefully, with a view to extract as much biological information as possible. Throughout this work I describe the development of open source computational tools for the analysis and visualisation of CLIP data. A computational pipeline based on hierarchical pre-mapping steps enables accurate quantification of non-coding RNAs from individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) datasets. Using the pipeline I describe novel tRNA binding for the DEAH-box helicase DDX3X and identify widespread binding of NSun2 and Trmt2A to pre-tRNAs. In collaboration with the lab of Prof. Folkert van Werven, I integrate m6A miCLIP with m6A-reader protein iCLIP data, alongside functional datasets in WT and methyltransferase deletion conditions in order to uncover the role of m6A in early budding yeast meiosis. Surprisingly, we find that the sole yeast m6A-binding protein, Pho92p, binds in both an m6A-dependent and an m6A-independent manner. m6A-dependent Pho92p binding partners are implicated in mRNA decay coupled to translation. Taken together I present powerful computational tools that will be of use to the wider community, alongside the interesting biological insights they have already enabled.

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