RNA regulatory networks in motor neuron development and function

 描述(申请人提供)：运动神经元发育和功能中的RNA调控网络在RNA水平上的转录后调控，如选择性剪接，在哺乳动物神经细胞在神经元发育过程中细胞和功能复杂性的产生中发挥关键作用。这种调节是由RNA结合蛋白(RBPs)与其目标转录本相互作用所决定的，从而深刻地影响 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。尽管在过去的几年里取得了非常显著的进展，但目前解剖神经元RNA调控网络的努力面临着两大挑战：i)作为调控网络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；ii)对于已发现的网络的功能评估至关重要的多个调控因子的同时扰动的困难和低效。这两个挑战都反映在对RBFox RBP家族的研究中，其中三个功能冗余的成员RBFOX1(A2BP1)、RBFox2(Rbm9)和Rbfox3(Neun)在许多类型的有丝分裂后神经元中优先表达，并被认为调节着大量重要的神经元转录本。到目前为止，只有一小部分RBFox靶向转录本在生理环境中得到验证，并且对RBFox蛋白或其靶向在特定神经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，它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，我们将同时使用体外但与生理相关的干细胞分化系统和体内小鼠模型，在该模型中，RBFox家族成员的各种组合在运动神经元中被耗尽，作为在分子和细胞水平上阐明其细胞类型特定功能的手段。由不同的RBFox家族成员直接调控的目标网络将通过全基因组的比较分析、特定细胞类型的高分辨率转录图谱、蛋白质-RNA相互作用的无偏图以及这些基因组和生化分析产生的多种数据的综合建模来确定。将对选定的RBFox靶子集进行功能验证，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RBPs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，而且有可能扩大我们对运动神经元疾病中RNA代谢紊乱的后果的理解。

 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.