Resumen de Reconstitution of fmrp-mediated mrna transport system in vitro
Learning and memory formation are based on the mechanisms of synaptic plasticity. Synaptic plasticity modulates development and strengthening of new neuronal connections, or synapses, upon learning and is based on local protein synthesis next to activated neuronal spines. Thousands of mRNA species are transported from the soma of neuron to dendrites, in order to be translated on demand. The mechanisms of this mRNA transport remain poorly understood.
Mutations of Fragile X-mental retardation protein (FMRP) cause a spectrum of mental retardation disorders. Among other functions, FMRP mediates signal-mediated mRNA transport and local translation in dendrites. In multiple attempts to understand how FMRP is implicated in mRNA transport, there were identified few motor protein candidates. Besides controversy in the literature, none of these motor proteins was demonstrated to bind directly and transport FMRP. In my PhD studies, I employed in vitro reconstitution assays, coupled to Total Internal Reflection (TIRF) microscopy, to test, which of the proposed candidate motors can transport FMRP along the microtubules and whether FMRP can co-transport mRNA molecules.
In order to understand the biochemistry of FMRP-mediated mRNA transport, I have purified and tested motor proteins from three Kinesin subfamilies. In this PhD thesis, I am reporting that FMRP binds directly to and is transported by Kinesin-2 motor (KIF3A/C heterodimer), but not by the other tested motors. Mutational analysis of FMRP suggests that its C-terminal region plays the biggest role in Kinesin-2 binding, and that this interaction does not depend on the RGG box region, known to recognise the G-quadruplex structure of FMRP’s mRNA targets. These results suggest that mRNA and motor binding by FMRP are not mutually exclusive, and thus FMRP must be capable to mediate mRNA transport. I also show that KIF3A/C motor binds several mRNA targets, with and without G-quadruplex structure, and that G-quadruplex mRNA competes with FMRP for motor binding. These results raise many questions that I address in the Discussion part of the thesis.
This work is the first of its kind, to my knowledge, to systematically test kinesin motor proteins for direct interaction with FMRP and to reconstruct an FMRP transport complex. I conclude that FMRP binds directly to the Kinesin-2 motor and that this complex moves processively along the microtubules. This complex is still missing its cargo, mRNA, which will be investigated beyond the scope of this PhD thesis. I analyse the speeds of used kinesin motors and compare them to the literature. In the end, I discuss possible reasons why FMRP was not binding the G-quadruplex mRNA in my experimental conditions and outline the caveats of in vitro reconstitution assays.
