Molecular mechanisms regulating mRNA transport and local translation in neurons.

The developing human brain must create short- and long-distance connections between dendrites and axons in a highly regulated fashion. The creation of new dendrites and the development and differentiation of synapses are an essential part of this process and require, in turn, the local translation of a large set of mRNAs. There is growing evidence that up to thousands of mRNAs are differentially translated in the dendritic compartment, thus making local translation a general and key process in neuronal development. Mistakes in the regulation of local mRNA translation lead to a range of developmental diseases and neuro-pathologies.
Local mRNA translation requires the selective transport and locally regulated translation of the mRNAs and is mediated by multi-functional, multi-domain RNA-binding proteins that recognise sequences or structures in the mRNAs and act as adaptors for the molecular motors and the cellular degradation and translation machineries. Despite several of these proteins having been identified, we know very little of how they target the cognate mRNAs and interact with the cellular machineries at the molecular level. Also we know very little of how these proteins are regulated by specific signals.

We work on IGF2 mRNA Binding Protein 1 (IMP1, also called Zipcode Binding Protein 1, ZBP1) as a paradigm for the RNA-binding proteins regulating local mRNA translation. IMP1 is a multi-functional, multi-domain RNA-binding protein that plays a key role in defining synaptic morphology in neurons and has a general function in regulating cell motility and differentiation. Functional information in fly, worm, chicken and mammals has shown that IMP1 regulates the transport and local translation of a number of different mRNAs, and has linked the protein to the transport of specific mRNAs (e.g. beta-actin) and to a well-defined regulatory mechanism that promotes local mRNA translation in response to signalling in neurons.
The questions we are asking are how IMP1 and other protein regulators recognise a diverse set of RNA targets, and how RNA recognition is linked to mRNA transport. We want to know how the RNA-binding proteins interact with the cellular mRNA transport machineries and how their functions are regulated by signalling at the molecular level. In the longer term, we want to obtain a broader understanding of the function of these proteins in local mRNA translation, that include their capability to localizing multiple, functionally related, targets, as a prelude to study the synergies between the locally translated proteins.

We will use structural and biophysical techniques to answer these questions in the IMP1 system and determine the molecular rules of IMP1 target recognition and of its regulation by signalling. Further, we will characterise IMP1 protein and RNA partners in neurons and use in cell transcriptome-wide assays to look at how IMP1 achieves the selection of the RNA targets in the cell, and to understand the RNA binding and re-modelling of the RNA is linked to the functional output. This work will provide a unique structural and molecular analysis of the functional interactions mediating local mRNA translation in mammals. The output will be used to inform the investigation of the function of IMP1 in processes linked to neuronal development and function, for example in dendritic arborisation and in the changes in synaptic morphology. Importantly, our understanding of IMP1 will provide tools and concepts for investigating other RNA-binding proteins with a vital role in neuronal functioning (e.g. Syncrip, FMRP and TDP43) that are linked to widespread and severe neuro-pathologies.

Dendritic arborisation and the development and differentiation of synapses are an essential part of neuronal development and require the local translation of a large ensemble of specific mRNAs. IGF2 mRNA binding protein 1 (IMP1) plays a key role in defining synaptic morphology in human neurons by regulating local mRNA translation. The goal of the proposed research is to understand, at a molecular level, how IMP1 regulates the transport and local translation of mRNAs in neurons.

We propose to:

- Determine how IMP1 recognize different RNA targets at the atomic level using NMR and crystallography. Then model the multi-step binding reactions using kinetic data from BLI experiments. Finally, understand how IMP1 uses different combinations of its RNA binding domains to select distinct sets of RNA targets in the cell using in parallel iCLIP assays.

- Explain how Src-mediated phosphorylation of IMP1, leads to the release and translation of the bound beta-actin mRNA by determining the changes in the structure and RNA binding properties of the protein and the recruitment of protein coregulators. Assess the specificity of Src-mediated IMP1 phospho-regulation.

- Directly link IMP1RNA-binding mode to IMP1-mediated mRNA localisation in neurons using smRNAFISH IF. Obtain a transcriptome-wide vision of IMP1-mediated mRNAs transport using MERFISH.

- Define the structural basis of the IMP1 interaction with the molecular motor KIF11 and the relation between IMP1-RNA interaction, IMP1-motor interaction and motor processivity, as assessed by single-molecule and bulk biophysics.

This work will result in novel tools and concepts useful to the study of the RNA-binding proteins regulating mRNA transport and local translation.

Fit with MRC priorities: The work will explain the molecular basis of the interaction between a key regulator of mRNA local translation and its RNA and protein targets, and how these interactions are regulated by specific signals. This process is key for the development of synaptic morphology and for dendritic arborisations and will further our knowledge in a key molecular neurologic mechanism, a key MRC priority. Also, IMP1-mediated regulation is a fundamental cellular mechanism that occurs in other proliferating cell types and its understanding will connect related processes in brain and other body systems, a second research priority for MRC. More broadly, this proposal has strategic relevance to the MRC's neurodegeneration programme. The concepts and experimental approaches described in this proposal can be applied to probe the molecular recognition processes underlying the function of proteins responsible for untreatable and devastating disorders linked to RNA metabolism (e.g. ALS / FTLD, in which the major hallmark pathology is deregulation of a multifunctional RNA binding protein called TDP43). I plan to explore this in collaboration with Rickie Patani and Jernej Ule.

Public Sector: The almost complete lack of disease modifying therapies for most neurodegenerative disorders and suboptimal therapies for some cancers are, at least in part, due to a lack of molecular understanding of the underlying disease mechanisms. Improving the outcome and efficiency of the treatment of neuropathologies and cancer is currently a challenge for the NHS, which is struggling to cope with an ageing population. The project has the potential to deliver a molecular understanding of a key regulatory process involved in neuronal physiology and cancer cell invasiveness. In the medium term (~10 years), this molecular understanding may result in the delivery of small molecules to control cancer metastasis and neuronal health.

General public: A better understanding of the regulation of IMP1 and of its paralogues may guide strategies to favour re-balancing and promoting plasticity of brain stem cell populations during ageing, and be of general benefit to the general population. The concepts / experimental approaches that will be explored here are applicable to a broad range of RNA binding proteins, which are increasingly implicated in neurodevelopmental disorders e.g (e.g. FMRP and Syncrip proteins). This proposal therefore has significant amplification potential.

Societal Impact: We expect the outcome of this research to have an impact on our understanding of the mechanisms underlying the development and function of neurons. This impact will be manifested not only by potentially guiding intervention strategies, but also by highlighting the role of local mRNA translation in a person's neuronal development. This, may also help promote further studies of the mechanisms regulating homeostasis at the synapse, also a key process in neuronal health where local translation of enriched mRNAs plays an important role.

Training and skills development: The work described in this proposal will directly result in the training of two PDRAs in, respectively, state-of-the-art genomic and imaging techniques and cutting-edge NMR and biophysical techniques. Importantly, the collaborative, multi-disciplinary interaction between the collaborating groups and PDRAs will expose all the group members to new ideas and approaches, and will be particularly beneficial to the PhD, master and undergraduate students in the groups, further amplifying the training and development benefits.