Understanding the Role of CLP1 in Mammalian mRNA Transcription and Cleavage

项目摘要/摘要 桥小脑发育不良(PCH)是由以下原因引起的儿童神经退行性疾病的集合 调节信使核糖核酸的处理或功能的基因突变。PCH亚型10(PCH10)由以下原因引起 RNA3‘端加工成员RNA激酶CLP1中的p.R140H纯合子突变 机械设备。CLP1在mRNA 3‘端加工中的作用及其病理生理机制 PCH10中的P.R140H仍然未知。 先前对PCH10患者来源的成纤维细胞和诱导神经元的研究表明，CLP1的功能可能是 在某些细胞类型中是关键的，但在其他类型中不是。运动神经元疾病是PCH10的一种穿透性表型，表明 运动神经元特别容易失去CLP1的活性或功能。我进行了RNA测序 CLP1p.R140H和CLP1基因敲除人类运动神经元并鉴定替代基因的不同特征 多聚腺苷酸化提示这些突变体可能通过不同的病理生理机制起作用。 这是由于失去了蛋白表达，而不是蛋白的活性。此外，一种纯合子的小鼠模型 CLP1P.K127A是一种蛋白激酶缺陷型突变体，表现出严重的进行性脊髓运动神经元变性， 但是，关于运动神经元中这种变异体的信使核糖核酸的处理还没有一个完整的特征。 已执行。由于变性是CLP1P.R140H和P.K127A一致的运动神经元表型 突变，I鉴定了带有p.R140H突变的人运动神经元的mRNA加工缺陷，即 预测干细胞来源的人运动神经元中CLP1激酶活性的扰动将改变mRNA3‘端 正在处理。基于这些发现，我推测CLP1激酶活性的丧失将影响 CLP1在人类运动神经元中的募集以转录基因和改变mRNA3‘端的处理。为了测试 这一假设，我将使用切割和运行和信使核糖核酸3‘端测序技术来解决以下问题 问题： 1)变异体如何影响CLP1在干细胞转录基因上的募集和分布 人类运动神经元的来源？ 2)CLP1p.K127A是否会改变干细胞来源的人类运动神经元的mRNA处理？

PROJECT SUMMARY/ABSTRACT Pontocerebellar hypoplasia (PCH) is a collection of pediatric neurodegenerative diseases caused by mutations in genes that regulate mRNA processing or function. PCH subtype 10 (PCH10) is caused by homozygous p.R140H mutation in the RNA kinase CLP1, a member of the mRNA 3’-end processing machinery. The function of CLP1 in mRNA 3’-end processing and the pathophysiological mechanism of CLP1 p.R140H in PCH10 remains unknown. Prior studies in PCH10 patient-derived fibroblasts and induced neurons suggest CLP1 function may be critical in certain cell types but not others. Motor neuron disease is a penetrant phenotype of PCH10, indicating motor neurons are particularly vulnerable to loss of CLP1 activity or function. I performed RNA sequencing of CLP1 p.R140H and CLP1 knockout human motor neurons and identified distinct signatures of alternative polyadenylation suggesting these mutants act through different pathophysiological mechanisms, potentially due to the loss of kinase activity rather than protein expression. Furthermore, a mouse model homozygous for CLP1 p.K127A, a kinase-deficient variant, exhibits severe and progressive spinal motor neuron degeneration, but a thorough characterization of mRNA processing in motor neurons with this variant has not been performed. Since degeneration is a consistent phenotype of motor neurons with CLP1 p.R140H and p.K127A mutation, and I identified mRNA processing defects in human motor neurons with the p.R140H mutation, I predict perturbation of CLP1 kinase activity in stem cell-derived human motor neurons will alter mRNA 3’-end processing. Based on these findings, I hypothesize that the loss of CLP1 kinase activity will affect the recruitment of CLP1 to transcribed genes and alter mRNA 3’-end processing in human motor neurons. To test this hypothesis, I will use CUT&RUN and mRNA 3’-end sequencing techniques to address the following questions: 1) How do variants affect the recruitment and distribution of CLP1 on transcribed genes in stem cell derived human motor neurons? 2) Does CLP1 p.K127A alter mRNA processing in stem cell derived human motor neurons?