Poster abstracts

Poster number 38 submitted by Jordan Farr

Pathogenic CLP1 p.R140H mutation alters mRNA processing in human motor neurons

Jordan S. Farr (Department of Genetics and Genome Sciences, Case Western Reserve University), Geneva R. LaForce (Department of Genetics and Genome Sciences, Case Western Reserve University), Cydni Akkesson (Department of Genetics and Genome Sciences, Case Western Reserve University), Eric J. Wagner (Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston), Ashleigh E. Schaffer (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract:
Pontocerebellar Hypoplasia Type 10 (PCH10) is a rare pediatric neurodegenerative disorder caused by bi-allelic p.R140H mutations in the multifunctional RNA kinase CLP11,2. The disease has been characterized by cerebellar and brainstem atrophy, progressive microcephaly, intractable tonic-clonic seizures, and progressive motor neuron loss1,2. However, new patients have revealed previously undocumented phenotypes while displaying little to no cerebellar involvement. A combination of work from our lab and others indicate that motor neuron degeneration is among the most penetrant phenotypes presented by mutations in CLP1, making the motor neuron a cell type of particular interest in the study of PCH103. To study the effects of the PCH10 mutation in motor neurons, we reprogrammed primary fibroblasts from an affected patient (p.R140H/p.R140H) and unaffected parental control (p.R140H/+) into induced pluripotent stem cells (iPSCs), then directed them to a motor neuron fate using an established differentiation protocol4. Because CLP1 is involved in both the processing of intron-containing tRNAs as well as 3’-end processing of mRNAs, we collected bulk RNA from the mature motor neurons and assayed for changes in mRNA and tRNA processing. Unlike reports documenting the PCH10 mutation in other cell types1-3, we find no tRNA processing defects in the affected motor neurons by Northern Blot. Instead, utilizing total RNA-seq and poly(A) Click-seq5, we have found broad, structural transcriptomic dysregulation. Affected motor neurons display a significant bias against the usage of intronic polyadenylation sites as well as a strong bias toward the expression of long genes, producing a gene expression signature that predicts neuronal hyperexcitability.

References:
1. Schaffer, A.E. et al. CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 157, 651-63 (2014).
2. Karaca, E. et al. Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function. Cell 157, 636-50 (2014).
3. Hanada, T. et al. CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 495, 474-80 (2013).
4. Martinez, F.J. et al. Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System. Neuron 92, 780-795 (2016).
5. Routh, A. et al. Poly(A)-ClickSeq: click-chemistry for next-generation 3-end sequencing without RNA enrichment or fragmentation. Nucleic Acids Res 45, e112 (2017).

Keywords: CLP1, motor neuron, intronic polyadenylation