Unravelling the physiological function of a highly conserved HNRNPA1 splice variant

揭示高度保守的 HNRNPA1 剪接变体的生理功能

• 批准号: RGPIN-2022-04268
• 负责人: VandeVelde, Christine
• 金额: $ 2.91万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761476
• 关键词: Unravelling; physiological; function; highly; conserved

My research program is dedicated to studying the molecular mechanisms that govern RNA binding protein (RBP) function in neurons, in both stressed and physiological conditions. Due to their highly polarized nature, neurons rely on numerous RBPs to support neuronal function, plasticity and survival. HNRNPA1 (heterogeneous ribonucleoprotein A1) encodes for two isoforms: hnRNP A1 and hnRNP A1B. These two proteins are identical, except for the inclusion of an additional 52 residues in the C-terminal glycine-rich domain in hnRNP A1B, owing to a highly conserved alternative splicing event. While hnRNP A1 is a well-known regulator of splicing, virtually nothing is known about the longer and phylogenetically ancestral isoform, hnRNP A1B. Using a custom antibody to selectively recognize hnRNP A1B, we have uncovered that hnRNP A1B is developmentally regulated such that its initial broad expression becomes restricted to motor neurons in adulthood. In addition, we have observed that unlike hnRNP A1, hnRNP A1B displays a granular cytoplasmic pattern in spinal motor neurons, in neurites of primary neurons and in a neuronal-like human cell line. We now have completed immunoprecipitation/mass spectrometry experiments to probe hnRNP A1B specific interactions in the CNS. These data indicate that hnRNP A1B may function in mRNA transport and local translational control, processes that are highly relevant to neurons. Moreover, we have observed that hnRNP A1B expression can be modulated by environmental stress, suggesting that it may play a role in the neuronal response to adverse conditions. As there are a limited number of motor proteins, but ~1500 RBPs, it is speculated that many of them function to improve transport specificity, serving as adaptors to link mRNAs with the transport machinery. Transported mRNAs are either translationally suppressed or co-translated following specific stimuli. Upon neuronal stimulation/activation (such as occurs in response to injury), distally-localized mRNAs are translated. Thus, RBPs effectively mediate spatial and temporal protein synthesis. The full repertoire of RBPs involved in these processes is not known. Our hypothesis is that hnRNP A1B plays a critical role in cytoplasmic RNA metabolic processes including axonal transport and localized translation that is essential for neuronal homeostasis in basal and stressed conditions. The aims are: Aim 1: Does hnRNP A1B contribute to the intracellular trafficking of mRNA in neurons? Aim 2: What is the function of hnRNP A1B in localized translation in neurons? Aim 3: Is hnRNP A1B upregulation a physiological response to insult/stimuli? Perspectives: This work will generate mechanistic insights on the physiological function of this understudied isoform in neurons and expand our knowledge of RBP regulation of neuronal response to insult. This information is critical to understand the diversity of neuronal functions and regulation of synaptic plasticity events.

我的研究计划致力于研究在应激和生理条件下控制神经元中RNA结合蛋白(RBP)功能的分子机制。由于其高度极化的性质，神经元依赖于大量的RBPs来支持神经元的功能、可塑性和生存。HnRNPA1(异质性核糖核蛋白A1)编码两种亚型：hnRNP A1和hnRNP A1B。这两种蛋白质是相同的，除了在hnRNP A1B的C端富含甘氨酸结构域中增加了52个残基，这是由于高度保守的选择性剪接事件。虽然hnRNP A1是众所周知的剪接调节因子，但几乎对更长的祖先亚型hnRNP A1B知之甚少。使用一种定制的抗体选择性地识别hnRNP A1B，我们发现hnRNP A1B受到发育调节，以至于其最初的广泛表达仅限于成年后的运动神经元。此外，我们还观察到，与hnRNP A1不同的是，hnRNP A1B在脊髓运动神经元、初级神经元的神经突起和神经元样的人类细胞系中显示出颗粒状的细胞质模式。我们现在已经完成了免疫沉淀/质谱学实验，以探测CNS中hnRNP A1B特异性的相互作用。这些数据表明，hnRNP A1B可能在mRNA转运和局部翻译控制中发挥功能，这些过程与神经元高度相关。此外，我们观察到hnRNP A1B的表达可以受到环境应激的调节，这表明它可能在神经元对不利条件的反应中发挥作用。由于马达蛋白的数量有限，但~1500个限制性商业惯例，推测其中许多蛋白具有提高运输特异性的功能，充当连接mRNAs与运输机械的接头。转运的mRNAs要么被翻译抑制，要么在特定刺激下共翻译。在神经元刺激/激活时(如对损伤的反应发生)，远端定位的mRNAs被翻译。因此，限制性商业惯例有效地调节了时空蛋白质的合成。这些进程中涉及的限制性商业惯例的全部内容尚不清楚。我们的假设是hnRNP A1B在细胞质RNA代谢过程中发挥关键作用，包括轴突运输和局部翻译，这对基础和应激条件下的神经元动态平衡是必不可少的。目的是：目标1：hnRNP A1B是否有助于神经元内信使核糖核酸的运输？目的2：hnRNP A1B在神经元定位翻译中的作用是什么？目的3：hnRNP A1B上调是否是对侮辱/刺激的生理反应？观点：这项工作将产生对神经元中这种未被研究的异构体的生理功能的机械性见解，并扩大我们对RBP调节神经元对侮辱的反应的知识。这些信息对于理解神经元功能的多样性和突触可塑性事件的调节至关重要。