Abstract
Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1 R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1 R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.
Highlights
IntroductionHomozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease
Homozygous mutation of the RNA kinase CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease
In conjunction with studies showing that CLP1 and PCF11 levels vary between tissues [17, 18], these findings suggest that cleavage factor II (CFII) may not be universally required for pre-mRNA cleavage, but rather contribute to cleavage in a transcriptand/or cell-type specific manner
Summary
Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. PAS usage was shifted from proximal to distal sites in the mutant mouse, in short and closely spaced genes Many such genes were expressed at lower levels in the Clp1R140H/− mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are dependent upon CLP1 for proper premRNA cleavage, suggesting that impaired mRNA 3′ processing may contribute to pathogenesis in PCH10. CLP1 has been hypothesized to stabilize the TSEN complex and facilitate intron excision [4] Consistent with this hypothesis, cleavage activity on an exogenous substrate was impaired in nuclear extracts from Clp1K127A/K127A (kinase-deficient) mouse and CLP1R140H/R140H human fibroblasts [1, 4]; cleavage of a similar pre-tRNA by the core TSEN complex was not improved by the addition of recombinant CLP1 [9]. The TSEN complex—the core components of which are TSEN2, TSEN34, TSEN54, and TSEN15—initiates tRNA splicing in the minority of Significance
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