Exploring the regulation of mRNA localisation and protein synthesis by RNA-binding proteins during angiogenesis

Abstract

Angiogenesis requires the tightly coordinated migration of endothelial cells (ECs) to form new vessels from pre-existing vasculature. Leading tip cells undergo delicate morphological changes which are underpinned by localised activity of proteins implicated in diverse signalling, migration and cytoskeleton rearrangement at the cell front. The subcellular distribution and local translation of messenger (m)RNAs is crucial to establish distinct functional domains in migrating cells, therefore research elucidating the post-transcriptional regulation of mRNA by RNA-binding proteins (RBPs) at the leading edge of migrating ECs is key to unravelling mechanisms of EC morphologies during blood vessel formation.

My research presented in this thesis provides an insight into how a novel RNA-interacting protein, AKAP12, and a well-known RBP, YB1, contribute to EC morphology during angiogenesis and elucidates their roles in regulating transcripts important for cell migration. Firstly, I evaluated the role of these proteins in small interfering (si)RNA-mediated loss-of-function angiogenesis assays. Here, I revealed that AKAP12 controls EC sprouting, as well as intricate filopodia dynamics; whilst YB1 orchestrates endothelial network formation and angiogenic sprouting. Next, I profiled their mRNA interactors enriched at the front of migrating ECs and identified that AKAP12 interacts with an mRNA encoding ABL2, a non-receptor tyrosine kinase implicated in remodelling of the actin cytoskeleton, and YB1 binds to many transcripts involved in cell migration, translation and cytoskeleton dynamics localised to the cell front. Lastly, I investigated how AKAP12 post-transcriptionally regulates ABL2 mRNA and reported for the first time a novel role for this protein in modulating the localisation of this mRNA to the plasma membrane as well as controlling its translation efficiency. Additionally, I showed that YB1:mRNA binding capabilities are regulated by AKT-activated VEGF stimulation and are altered in protrusions of migrating ECs. In preliminary studies, I also reported that translation of ACTB, the transcript encoding β-actin, is regulated by YB1 through AKT activation downstream of VEGF.

With this, I proposed two models: 1) AKAP12 is targeted to the plasma membrane, anchors ABL2 mRNA and ensures the efficiency of its translation where both AKAP12 and ABL2 regulate EC morphology during angiogenic sprouting; 2) upon VEGF-A stimulation, AKT-mediated phosphorylation of YB1 releases bound mRNAs for translation where nascent local proteins take part in cytoskeleton dynamics and EC sprouting. Together, reaching a better understanding of regulatory functions and mechanisms of RBPs in the context of angiogenesis may lead to potential therapeutic targets for angiogenic-related diseases.

Thesis is embargoed until 31st December 2026.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Guilherme Costa (Supervisor) & Tim Curtis (Supervisor)