Understanding the Role of CLP1 in Mammalian mRNA Transcription and Cleavage

项目总结/摘要 脑桥小脑发育不全（PCH）是一组儿童神经退行性疾病， 调节mRNA加工或功能的基因突变。PCH亚型10（PCH 10）是由 RNA激酶CLP 1（mRNA 3 '端加工成员）中的纯合p.R140H突变 机械. CLP 1在mRNA 3 '端加工中的作用及其病理生理机制 PCH 10中的p.R140H仍然未知。 先前在PCH 10患者来源的成纤维细胞和诱导的神经元中的研究表明，CLP 1功能可能是 在某些细胞类型中至关重要，但在其他细胞类型中并非如此。运动神经元疾病是PCH 10的一种外显表型，表明 运动神经元特别容易丧失CLP 1活性或功能。我进行了RNA测序， CLP 1 p.R140H和CLP 1敲除人运动神经元，并鉴定了替代性神经元的不同特征。 多聚腺苷酸化表明这些突变体通过不同的病理生理机制起作用， 这是由于激酶活性的丧失而不是蛋白质表达的丧失。此外，一种纯合的小鼠模型， CLP 1 p.K127A是一种激酶缺陷型变体，表现出严重且进行性的脊髓运动神经元变性， 但是对于这种变异体在运动神经元中的mRNA加工过程的全面表征还没有被 执行。由于退行性变是具有CLP 1 p.R140H和p.K127A的运动神经元的一致表型， 突变，我确定了mRNA加工缺陷，在人类运动神经元与p.R140H突变，我 预测干细胞衍生的人类运动神经元中CLP 1激酶活性的扰动将改变mRNA 3 '端 处理.基于这些发现，我假设CLP 1激酶活性的丧失将影响 在人类运动神经元中，CLP 1向转录基因的募集并改变mRNA 3 '末端加工。测试 基于这一假设，我将使用CUT&RUN和mRNA 3 '端测序技术来解决以下问题 问题： 1)变异体如何影响CLP 1在干细胞转录基因上的募集和分布 衍生的人类运动神经元 2)CLP 1 p.K127A是否改变干细胞衍生的人运动神经元的mRNA加工？

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?