Transcription, Trafficking, Translation: dissecting the spatiotemporal mechanisms underlying localised protein synthesis. (360G-Wellcome-110170_Z_15_Z)

Transcription, Trafficking, Translation: dissecting the spatiotemporal mechanisms underlying localised protein synthesis We build this proposal around the hypothesis that in the majority of cells mRNAs are spatially located and translated in a highly controlled manner involving the interaction of cis- and trans- acting factors with RNA-binding proteins (RNA-BPs). We also hypothesise that the systems involved in RNA localization respond to perturbation to post-transcriptionally regulate gene e xpression. The synthesis of many proteins occurs in a spatially segregated manner that involves positioning of mRNAs to the precise cellular location where the corresponding protein products are required. This process is used in highly polarised and asymmetric cells as a way of restricting protein synthesis to specific, specialised compartments including in D. melanogaster embryos, neurons, budding yeast and differentiating cells [1-5]. However, the information gained from studies which fo cus on polarized cells/or very defined developmental systems, is rather limited [1] since recent system level analyses demonstrate that spatially regulated translation coupled to mRNA localization is a general occurrence in less polarised cells. For example, mRNAs encoding components of peroxisomes [10], ER and mitochondria [11, 12] associate with, and are translated at, the membranes of these organelles. To date, there is little information about the spatial organisation of the translation of proteins that are translated in the cytosol or peripherally associated membrane proteins [13-15]. Thus despite recent advances, many of the underlying mechanism(s) controlling temporal and spatial regulation of gene expression in cells that do not show a high degree of polarisation remain unknown. Three major mechanisms have been proposed to contribute to mRNA trafficking: diffusion and cytoplasmic streaming, asymmetric degradation and active transport. The latter involves microtubules or actin filaments forming super-highways for transport mediated by kinesins, dyneins and myosins [6, 7]; after transport mRNAs are tethered to specific subcellular niches permitting spatially defined translation [8, 9]. Importantly, in this proposal we are not aiming to study the direct transport of mRNA along microtubules and actin filaments, instead we will interrogate the steady state location and translation of the transcriptome. We will combine and further develop complementary technologie s to ask three related questions, each addressing a different critical point of localised translation that is currently poorly understood: i) What are the commonalities and the distinctions between the mechanisms that allow localization and translation of mRNAs at specific subcellular locations and structures? The processes that lead to mRNA localization and localized translation are complex and highly coordinated [1, 6, 7] mediated by the interaction of mRNAs with their cognate RNA-BPs; generally through recognition of cis-acting regulatory elements [16]. However, understanding how RNA-BPs promote localization and translation of multiple mRNAs is challenging, since thousands of mRNAs are localized, yet only 600-800 mammalian RNA-BPs have been identified [17]. Thus w