Unveiling the role of RAB13 localisation in angiogenesis

Deciphering the complex biology of cell migration is key to understand human-related disorders. During angiogenesis, endothelial cells engage in coordinated migration events to form new blood vessels from parental counterparts. However, while subcellular localisation of mRNAs and localised translation are fundamental steps between gene transcription and protein activity, whether local regulation of gene expression controls the complex morphogenetic process of angiogenesis has never been addressed. Here, we focused on RAB13 mRNA, a transcript enriched at the leading front of many migratory cell types. We set out to delete regions in the 3'UTR of RAB13 mRNA responsible for its subcellular distribution in vitro and in vivo and to study its localised role during angiogenesis. After identifying regions responsible for the localisation of RAB13 mRNA, we used CRISPR/Cas9 technology with gRNAs targeting the 3'UTR of both human and zebrafish orthologues. We performed RNA-seq experiments using RNA derived from control and mutant samples to 1) investigate whether potential off-target sites were edited by CRISPR/Cas9 and 2) analyse the expression of potential RAB13 splicing variants. Samples analysed: HUVECs - 1x Wild type and 1x Homozygous RAB13 3'UTR; Zebrafish - 2x Wild type and 2x Homozygous RAB13 3'UTR. Description of RAB13 3'UTR mutations: Deletion of 190 nucleotides from Human RAB13 3'UTR Deletion of 482 nucleotides from Zebrafish RAB13 3'UTR