In vitro reconstitution of kinesin-based mRNA transport
Cytoplasmic mRNA transport along microtubules is an essential mechanism of symmetry breaking, observed in a multitude of biological systems. In neurons, delivery of mRNAs to remote dendritic and axonal locations is essential for neuronal differentiation, axon guidance and synaptic plasticity. Many proteins were identified that play a role in neuronal mRNA transport but the essential factors required to transport any mRNA along microtubules remained unknown. While our laboratory is interested in dissecting the mechanochemistry generating distribution patterns of 1000s of mRNAs, we focussed in this study on revealing the first essential set of building blocks required to transport an axonally localised mRNA. We use an in vitro reconstitution approach coupled with Total-Internal-Reflection-Microscopy (TIRF-M) to identify a minimal system capable of transporting an mRNA. We expressed all required full-length proteins recombinantly and in vitro transcribed required RNA fragments. With in-vitro motility assays, we show that a kinesin motor can transport our candidate RNA binding protein (RBP) along microtubules. We found that this requires an additional adaptor protein. By Microscale-Thermophoresis (MST), we further show that our candidate RBP is capable of binding the target-mRNA 3’UTR and the adaptor protein with nM-range affinities. Finally, we demonstrate that these interactions are of sufficient strength to drive processive mRNA transport in vitro. Our results reveal for the first time a minimal set of 3 proteins sufficient to drive processive, kinesin-based mRNA transport.
CRG Center for Genomic Regulation, Barcelona, Spain
Domain 1 - UMR 3348 - Genotoxic Stress and Cancer