RNA regulatory networks in motor neuron development and function

 运动神经元发育和功能中的RNA调控网络RNA水平的转录后调控，如选择性剪接，在神经元发育期间哺乳动物神经细胞的细胞和功能复杂性的产生中起关键作用。这种调节是由RNA结合蛋白（RBP）与其靶转录本相互作用决定的，从而深刻地影响了细胞的功能。 转录组的输出。该项目的长期目标是阐明这些RNA调控网络的组织原理及其在系统水平上对神经元发育的功能影响。尽管在过去几年中取得了非常显著的进展，目前解剖神经元RNA调控网络的努力面临着两个主要挑战：i）用作调控网络的基因组和生化分析材料的主要来源的脑组织的细胞异质性，以及ii）对于所发现的网络的功能评估重要的多个调控因子的同时扰动的困难和效率低下。这两个挑战都反映在Rbfox RBP家族的研究中，其中三个功能冗余成员Rbfox 1（A2 bp 1），Rbfox 2（Rbm 9）和Rbfox 3（NeuN）优先在许多类型的有丝分裂后神经元中表达，并被认为调节大量重要的神经元转录本。到目前为止，只有少量的Rbfox靶转录本在生理环境中得到验证，并且Rbfox蛋白或其靶在特定神经元细胞类型中的功能知之甚少。在这项提案中，我们将特别关注运动神经元，这是肌肉收缩和运动所需的神经细胞，在几种致命的神经退行性疾病中丢失。我们假设Rbfox蛋白的协同作用对运动神经元的发育和功能至关重要。为了验证这一假设，我们将采用，在平行的，在体外，但生理相关的干细胞分化系统和体内小鼠模型，其中各种组合的Rbfox家族成员被耗尽在运动神经元作为一种手段，阐明其细胞类型特异性功能的分子和细胞水平。由不同的Rbfox家族成员直接调节的靶网络将通过全基因组的比较分析来定义，细胞类型特异性转录组的高分辨率分析，蛋白质-RNA相互作用的无偏地图，以及这些基因组和生化测定产生的多种数据模式的综合建模。将对Rbfox靶点的选定子集进行功能验证，以建立剪接变体与神经元发育的特定方面之间的联系。我们的研究结果不仅将为神经元RBP在运动神经元生物学中的功能和潜在的分子机制提供新的见解，而且还可能扩大我们对运动神经元疾病中RNA代谢中断的后果的理解。

 DESCRIPTION (provided by applicant): Project Summary 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.