Non-redundant functions of Hu proteins as neuron-specific splicing regulators

描述（由申请人提供）：我正在研究Hu蛋白家族成员作为I型神经纤维瘤病（NF 1）外显子23 a选择性剪接的负调节因子具有非冗余功能的假设。这项研究将有助于确定这些剪接抑制因子在功能上如何分化的机制，也将揭示Hu蛋白调节剪接的机制，这一点还不清楚。初步数据表明，HuC是一个强抑制因子，HuB是一个弱抑制因子，NF 1外显子23 a包含。在我的项目中，我将调查是否HuB和HuC在调节小鼠小脑颗粒神经元内源性NF1剪接的能力不同。此外，我将通过体外剪接和剪接体组装试验研究HuB和HuC在剪接抑制剂效力方面差异的机制。我将使用一种新的系统进行这些试验，该系统涉及从分化为神经元的小鼠胚胎干细胞而不是从含有细胞类型异质混合物的永生化细胞系或整个组织制备核提取物。公共卫生相关性：选择性剪接是一个对神经系统内的许多功能（如神经元发育）至关重要的过程，选择性剪接中的错误可能导致疾病，因此了解该过程是如何调节的非常重要。我的研究将有助于阐明Hu蛋白家族调节I型神经纤维瘤病（NF1）外显子23a包含的机制，这是一个对NF1功能很重要的剪接事件，可能会影响NF1疾病的严重程度。此外，我的研究将增加对Hu蛋白分子功能的理解，Hu蛋白与一种称为副肿瘤综合征的疾病有关。

DESCRIPTION (provided by applicant): I am investigating the hypothesis that Hu protein family members have non-redundant functions as negative regulators of the alternative splicing of neurofibromatosis type I (NF1) exon 23a. This research will help to determine the mechanisms behind how these splicing suppressors have diverged in function, and will also shed light on the mechanisms by which Hu proteins regulate splicing, which is not well understood. Preliminary data indicate that HuC is a strong suppressor and HuB a weak suppressor of NF1 exon 23a inclusion. In my project, I will investigate whether HuB and HuC vary in ability to regulate endogenous NF1 splicing in primary mouse cerebellar granule neurons. In addition, I will investigate the mechanism of the difference between HuB and HuC in splicing suppressor potency by performing in vitro splicing and spliceosome assembly assays. I will perform these assays using a novel system involving preparation of nuclear extract from mouse embryonic stem cells differentiated into neurons rather than from immortalized cell lines or whole tissues containing a heterogeneous mixture of cell types. Public Health Relevance: Alternative splicing is a process that is critical for many functions within the nervous system, such as neuronal development, and errors in alternative splicing can lead to disease, making it important to understand how the process is regulated. My study will help to elucidate the mechanism by which the Hu protein family regulates neurofibromatosis type I (NF1) exon 23a inclusion, a splicing event that is important for NF1 function and could possibly affect severity of NF1 disease. In addition, my studies will increase understanding of the molecular functions of the Hu proteins, which are associated with a disease called paraneoplastic syndrome.