RNA regulatory networks in motor neuron development and function Post-transcriptional regulation at the RNA level, such as alternative splicing, plays a critical role in generating of the cellular and functional complexityof mammalian nerve cells during neuronal development. This regulation is dictated by RNA-binding proteins (RBPs) interacting with their target transcripts, thereby profoundly affecting the output of the transcriptome. The long-term goal of this project is to elucidate the organizational principles of these RNA regulatory networks and their functional impact on neuronal development at the systems level. Despite very significant progress made over the past few years, current efforts to dissect neuronal RNA regulatory networks are facing two major challenges: i) cellular heterogeneity of the brain tissue used as a major source of material for genomic and biochemical analysis of regulatory networks, and ii) difficulty and inefficiency of simultaneous perturbation of multiple regulators important for functional evaluation of the discovered networks. Both challenges are reflected in studies of the Rbfox RBP family, in which the three functionally redundant members Rbfox1 (A2bp1), Rbfox2 (Rbm9) and Rbfox3 (NeuN) are preferentially expressed in many types post-mitotic neurons and are believed to regulate a large set of important neuronal transcripts. So far, only a small number of Rbfox target transcripts have been validated in physiological contexts and the function of Rbfox proteins or their targets in specific neuronal cell types is poorly understood. In this proposal, we will particularly focus on motor neurons, which are the nerve cells required for muscle contraction and movement and are lost in several fatal neurodegenerative diseases. We hypothesize that a concerted action of Rbfox proteins is critical for motor neuron development and function. To test this hypothesis, we will employ, in parallel, an in vitro but physiologically relevant stem cell differentiation system and an in vivo mouse model, in which various combinations of Rbfox family members are depleted in motor neurons as a means of elucidating their cell type-specific function at the molecular and cellular levels. The target networks directly regulated by different Rbfox family members will be defined by comparative analysis of genome-wide, high-resolution profiling of cell type-specific transcriptomes, unbiased maps of protein-RNA interactions, and integrative modeling of multiple modalities of data generated by these genomic and biochemical assays. Functional validation of a select subset of Rbfox targets will be performed to establish the link between splice variants and specific aspects of neuronal developments. Results from our studies will not only provide novel insights into the function of neuronal RBPs in motor neuron biology and the underlying molecular mechanisms, but also have the potential to expand our understanding in the consequence of disrupted RNA metabolism in motor neuron diseases.
Post-transcriptional gene expression regulation through specific protein-RNA interactions is critical for expanding the complexity of the mammalian nervous system with implications in an expanding list of neuronal disorders. The proposed studies investigate RNA regulatory networks in development and function of spinal motor neurons. Information obtained in the proposed studies will provide insights into the fundamental mechanisms of post-transcriptional regulation and the functional consequences in a defined, clinically relevant neuronal cell type.
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