Understanding the biochemistry that drives mRNA transport
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. In neurites, 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 defined? Which mechanisms trigger assembly and disassembly of mRNA transport complexes?
Remarkably, mRNA transport is only one aspect of transcript-cytoskeleton interaction: different types of RNPs are transported along microtubules, interact with different MAPs that 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.
Our lab uses new approaches to understand the mechanochemistry driving differential mRNA distribution. We combine matrix-screening approaches, developed in our lab, with microscopy-coupled biochemical in vitro reconstitutions. The screen reveals direct interactions between microtubule-associated proteins (MAPs) and mRNP components while in vitro reconstitutions teach us how mRNP transport components dynamically interact to enable processive and selective mRNA transport.
For biophysical complex characterization, 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. This way, we obtain an understanding of the dynamics of mRNA transport complex assembly in conjunction with absolute numbers about complex stoichiometries and affinities within the complex.
In vitro reconstitution experiments allow us to understand the specific function of each building block in an mRNA transport complex. Combined with automated particle tracking of TIRF – microscopy data this approach provides sound statistics that help us to understand how different mRNAs are transported.
For instance, we recently discovered that the microtubule plus-end tracking protein APC selectively binds G-rich motifs found in mRNA 3’UTRs. We could reconstitute minimal RNPs in vitro, which are recruited to microtubules through APC’s microtubule-binding domains. APC RNPs diffuse on the microtubule lattice and can be picked-up for processive transport by the kinesin-2 KIF3AB. This in turn requires the cargo-adaptor protein KAP3 which links APC-RNPs to KIF3AB.
We also do a lot of experiments with single-molecule sensitivity. This allows us for example to understand the stoichiometry of reconstituted mRNA transport complexes. In the case of APC-RNPs we found that a single KIF3ABKAP3 heterotrimer mostly transports APC dimers which can bind one RNA molecule per APC monomer. Interestingly, APC also binds microtubules as dimer with a maximum of two RNAs under saturating RNA concentrations.
Discovery of direct interactions within RNPs and between RBPs and microtubule-binding proteins: Little is known about how membrane-free cargoes like mRNAs are coupled to motor proteins. 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.
Reconstitutions of different mRNA transport complexes: In neurons, different mRNAs apparently use different mRNA transport pathways. But which different mRNA transport pathways exist is not known. We use new interactions discovered by our screens to predict new mRNA transport pathways. After validation of new interactions by an independent assay, we assemble new mRNA transport modules from bottom-up in vitro to understand their function.
Transport dynamics in neurons: We use neurons induced from mouse embryonic stem cells to dissect the protein networks that control cellular mRNA logistics. To this end, we invest a lot of effort into techniques that allow fast perturbations in living cells. We believe that fast perturbations are essential to assess the function of mRNA transport building blocks without confounding effects from slower compensatory mechanisms.