Understanding the Role of CLP1 in Mammalian mRNA Transcription and Cleavage

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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?