Cytoplasmic mRNA transport along microtubules is an essential mechanism of symmetry breaking required for many biological processes as synaptic plasticity, axis determination during development and cell migration. The ability to control the protein configuration of different cell regions individually allows the cell to create domains with specialized functions. Prominent examples of cell regions for whose functionality RNA localization was shown to be crucial are axons and dendrites. Here the right localization of a certain RNA population is important for memory formation and maintenance while mutations affecting components of RNA transport systems lead to severe neurodegenerative diseases. Our laboratory is interested in the molecular mechanisms underlying cytoplasmic RNA transport along cytoskeletal elements, especially microtubules. Many unsolved questions surround cytoplasmic RNA transport which include: What is the minimal set of components needed to transport an RNA? How do motors select which RNA to transport? How is the amount of transported RNAs regulated?
Remarkably, mRNA transport is only one aspect of transcript-cytoskeleton interaction: different types of RNPs are transported along microtubules, interact with different MAPs which localise to different microtubule regions. Furthermore, MAPs were shown to directly bind to RNAs while canonical RBPs can bind to microtubules as well. The function of this crosstalk between RNA- and cytoskeleton-interactors is largely unknown.
In order to understand these mechanisms, we aim to uncover how the complex RNA distributions observed in cells are generated through the intrinsic properties of the involved factors. To this end, we employ a bottom-up approach which aims 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 single-molecule fluorescence microscopy assays, size-exclusion coupled multi-angle-light-scattering measurements (SEC-MALS) and microscale-thermophoresis measurements to obtain an understanding of the dynamics of mRNA transport complex assembly in conjunction with absolute numbers about complex stoichiometries and affinities within the complex.
– Detection of direct interactions within RNPs and between RNPs and microtubules: We created libraries of full-length 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 analyze the functionality of known components of RNA transport complexes in a quantitative manner.
– Dual function of RBPs in transport and translational regulation: We test the effect of pure RBPs on translation regulation in microfluidic coupled microscopy assays.
– Probing RNP composition ex-vivo: Single-molecule pulldowns from cell extracts are used to unravel stoichiometries and composition of neuronal RNPs.