Defining the role of mRNA localisation in cell migration

Abstract

Cells are organised so that specific functions are carried out in specific regions. The necessary components for these functions, including proteins, cytoskeleton, and organelles, must therefore be non-uniformly distributed in subcellular space. One way this can be achieved is by actively targeting messenger RNA to, and locally translating them within, sites where the encoded protein is required. However, the diversity of RNA subcellular localisations, the mechanisms by which they reach their destination, and the function that RNA localisation confers on translated proteins, particularly in the context of cell migration, is not well understood. Here, using RNA-seq coupled with a cell fractionation scheme, the cell-type diversity of RNA enrichment in migratory protrusions has been revealed. In doing so, groups of RNAs that display shared RNA localisation patterns were identified, including distally-located highly-polarised RNAs, and distinct peri-Golgi associated RNAs. Then, using motif-enrichment analysis in combination with the MS2-MCP reporter system for RNA visualisation, G-rich 3’UTR sequence motifs were shown to be the driving force behind RNA polarisation. Next, endogenous RNA localisation could be disrupted by genomic excision of G-rich sequence motifs using CRISPR-Cas9 editing tools, to provide novel insights into the roles of localised RNAs. RAB13 RNA localisation was revealed to define a zone of active filopodia production in the leading front of migratory endothelial cells, and that this is essential for directional pathfinding during in vivo angiogenesis. Finally, polarised TRAK2 RNA was then shown to be required to maintain normal mitochondria distribution in the leading edge, and for regulating endothelial cell motile speed during matrix migration. Together, this thesis has characterised the cell-type diversity of RNA localisation patterns in motile cells, shed light on the mechanism behind polarised RNA localisation, and greatly expanded the realm of known molecular functions of localised RNAs. Therefore, the evidence presented within contributes significantly to our understanding of how RNA localisation spatiotemporally regulates protein activity. Moreover, this work will have implications for future research concerning the molecular control of cell motility during development.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sam Griffiths-Jones (Supervisor), Patrick Caswell (Supervisor) & Shane Herbert (Supervisor)