Regulation of mRNA transport by multi-domain mRNA binding proteins

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Techniques used (unchanged):Bio-layer interferometry (BLI), isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, scaffold-independent analysis (SIA), small-angle x-ray scattering (SAXS), x-ray crystallography.RNA-binding proteins (RBPs) regulate gene expression in a spatial and temporal manner, by recruiting specific mRNAs to cell machinery involved in RNA processing, transport, degradation and translation. The RBP Syncrip, is an important regulator of embryonic neuronal development, and is an important system to understand the transport of localised mRNAs. Syncrip is known to bind a range of different RNA targets, and exert multiple functions, in a target-dependent manner. Dysfunction of Syncrip results in a number of neurological diseases. The protein consists of an N-terminal domain, which we have recently demonstrated to bind RNA and that we have named N-terminal Unit for RNA Recognition (NURR) and of three C-terminal RRM domains. Based on mRNA interactome capture data, all four domains are involved in mRNA recognition - albeit to different extents with each target. This is observed also for non-mRNA targets, for example, it has been shown that the NURR domain is the key domain for the interaction of hEXO motif on miRNAs that are loaded onto exosomes, and for exosomal loading. However, the three RRM domains also participate in the binding. We propose to examine how Syncrip utilises these domain to recognise specific mRNA targets to be transported to specific embryonic compartments. In this context, we will be looking at the recognition of specific RNA sequences by the RRM domains, at the role of the protein in packaging of the RNA molecule and at the protein's regulation by the phosphorylation - which connects RNA regulation by RNA-binding proteins to signalling networks. We will in parallel look at how the link between signalling and RNA recognition is mediated in other RNA-binding proteins.