Unravelling the physiological function of a highly conserved HNRNPA1 splice variant

我的研究项目致力于研究应激和生理条件下神经元中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rbp来支持神经元的功能、可塑性和存活。HNRNPA1（异质核糖核蛋白A1）编码两种亚型：hnRNP A1和hnRNP A1B。这两个蛋白是相同的，除了在hnRNP A1B的c端富含甘氨酸的区域中包含额外的52个残基，这是由于高度保守的选择性剪接事件。虽然hnRNP A1是一个众所周知的剪接调节因子，但实际上对更长的和系统发育上的祖先异构体hnRNP A1B一无所知。使用定制抗体选择性识别hnRNP A1B，我们发现hnRNP A1B受发育调节，因此其最初的广泛表达在成年期仅限于运动神经元。此外，我们还观察到，与hnRNP A1不同，hnRNP A1B在脊髓运动神经元、原代神经元的神经突和类似神经元的人类细胞系中表现出颗粒状的细胞质模式。我们现在已经完成了免疫沉淀/质谱实验来探测hnRNP A1B在中枢神经系统中的特异性相互作用。这些数据表明，hnRNP A1B可能在mRNA转运和局部翻译控制中起作用，这些过程与神经元高度相关。此外，我们观察到hnRNP A1B的表达可以被环境应激调节，这表明它可能在神经元对不利条件的反应中发挥作用。由于运动蛋白的数量有限，但rbp的数量约为1500个，因此推测其中许多具有提高转运特异性的功能，作为连接mrna与转运机制的适配器。转运mrna在特定刺激下被翻译抑制或共翻译。当神经元受到刺激/激活时（如对损伤的反应），远端定位的mrna被翻译。因此，rbp有效地介导了蛋白质的时空合成。参与这些过程的rbp的全部曲目尚不清楚。我们的假设是hnRNP A1B在细胞质RNA代谢过程中起关键作用，包括轴突转运和局部翻译，这对基础和应激条件下神经元的稳态至关重要。目的是：目的1:hnRNP A1B是否有助于神经元中mRNA的细胞内运输？目的2:hnRNP A1B在神经元局部翻译中的作用是什么？目的3:hnRNP A1B上调是对侮辱/刺激的生理反应吗？观点：这项工作将对这种未被研究的同种异构体在神经元中的生理功能产生机制见解，并扩大我们对RBP调节神经元对侮辱反应的认识。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。

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.