RNA localisation in endothelial cells: elucidating spatial regulation of gene expression to understand blood vessel growth and homeostasis

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Messenger (m)RNA molecules are the middlemen between genes and proteins. They are transcribed from DNA and then used as translation templates to synthetise functional proteins. Most mRNAs are not equally distributed throughout the cell. Depending on distinct regulatory factors, these molecules tend to accumulate within particular regions of the cell, generating subcellular patters of distribution. This phenomenon is part of a general mechanism called RNA localisation, which has been described in several cell types. Using key biological models, researchers have found that the localisation of mRNAs is fundamentally important for particular cellular functions and without it, cells may fail to respond correctly to stimuli, to acquire certain fates or even to be properly shaped. Hence, it is thought that RNA localisation has major implications in animal development and it can be the basis of some human disorders.Our lab found that RNA localisation takes place in endothelial cells, the building blocks that line all blood vessels. In this context, distribution of mRNAs to particular subcellular compartments, where they are translated, regulates how vessels grow. This work provided new evidence that the compartmentalisation of mRNAs is crucial for tissue formation. However, many questions remain unanswered. What are the exact molecular mechanisms responsible for the targeting of mRNAs in endothelial cells? Are mechanisms of localisation also implicated in other aspects of vascular function, such as the regulation of the barrier formed by the endothelial cells in fully formed vessels? In this proposal, we will use distinct models and methodologies to answer these questions. Our most recent findings leading to this proposal indicate that mRNAs can be bound by proteins that remodel the cytoskeleton, a structure that ensures cell architecture and behaviour. In particular, some of these are located to sites where cells adhere to the substrate or to each other. This suggests that such factors could have RNA localisation roles yet to be described. Furthermore, given their relevance in cell movement and cell-cell contacts, we hypothesise that cytoskeletal remodelling proteins distribute mRNAs during blood vessel formation and endothelial barrier function.To explore these ideas, we will first identify the exact mRNAs bound by the cytoskeletal remodelling proteins and then characterise their spatial co-distribution. We will deplete the proteins of interest from motile endothelial cells as they form new vessels and use state-of-the-art microscopy to analyse shifts in mRNA localisation patterns. We will also investigate if the loss of these proteins alters local target translation. Next, we plan to focus on the endothelial layer within blood vessels. For this, we will use extracellular factors that disrupt the barrier formed by endothelial layers and investigate if these alter the localisation of mRNAs. In addition, we will block the binding of the cytoskeletal remodelling proteins to their mRNA targets to prove that this process is implicated in endothelial barrier biology. Given that the work described so far takes advantage of in vitro tools mimicking vascular biology in a petri dish, in the final stages of the project we will transpose our tests to the mouse retina, an excellent tool to study blood vessel formation and maintenance in vivo. Such approach will be crucial to understand the importance of RNA localisation in the context of a complex tissue. We hope that this framework will help us to fully grasp how subcellular mRNA distribution determines cell behaviour and tissue biology. This will open avenues in precision medicine not only to treat vascular related pathologies, but also to target cell migration and tissue maintenance in other disorders.