Cytoskeleton dependent RNA distribution mechanisms

Cytoplasmic mRNA transport along microtubules is an essential mechanism of symmetry breaking required for many biological processes as synaptic plasticity and long term memory formation.

Our lab wants to understand how the complex cellular mRNA distributions, which are generated by active transport, are organised through the intrinsic properties of the involved factors. To this end we employ a bottom-up approach to reconstitute mRNA transport complexes and sub-complexes from purified, recombinant mouse proteins and in vitro transcribed mRNAs and mRNA-fragments. We combine data from ensemble and multi-colour single-molecule fluorescence microscopy assays, biophysical analytics and newly developed high-throughput protein-protein and protein-RNA interaction screens. Biochemical in vitro reconstitutions strategies have been used successfully in the past to unravel the functional principle of complex enzymatic machineries which includes cytoskeletal- and transport-systems. It is now timely to undertake a systematic bottom-up approach to answer the many fundamental questions unanswered in MT dependent neuronal mRNA transport systems: What is the minimal set of components needed to transport an mRNA along microtubules? Which principles determine number, species and destination of transported RNA? How is mRNA cargo release at the destination controlled? Which different mRNA transport pathways exist in mammalian neurons that give rise to the observed distribution patterns of mRNAs?

• Detection of direct interactions within RNPs and between RNPs and microtubules: We created libraries of full-lengths RNA binding proteins (RBPs) and microtubule associated proteins (MAPs) and develop new high throughput screening approaches for the detection of direct interactions between these proteins and these proteins & RNAs.
• RNP-motor coupling: We use interactions found by our new screens to reconstitute minimal kinesin and dynein-based RNA transport complexes. We use these reconstituted systems as experimental framework to analyse the functionality of known components of RNA transport complexes in a quantitative manner.

Yang JS, Garriga-Canut M, Link N, Carolis C, Broadbent K, Beltran-Sastre V, … Maurer SP.
“rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions.”
Nature Communications, 9(1): 3747, 2018. https://doi.org/10.1038/s41467-018-06128-x

Yang JS, Garriga-Canut M, Maurer SP.
“A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH.”
Protocol Exchange, 2018. https://doi.org/10.1038/protex.2018.112