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Clp1 knockdown lentigenic mouse generation

ABSTRACT: Clp1 knockdown lentigenic mouse generation

PROVIDER: PRJNA591193 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10517902.fastq.gz	Fastqsanger.gz
	SRR10517903.fastq.gz	Fastqsanger.gz

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Clp1 knockdown lentigenic mouse generation

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Project description:Homozygous mutation of the RNA kinase CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the 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 upregulated, and other fragments downregulated. However, the spatiotemporal patterns of dysregulation did not support a role in pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on pre-mRNA cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- 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 also 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 particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that the contribution of mRNA 3’ processing to pathogenesis in PCH10 merits further investigation.

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Project description:Schizosaccharomyces pombe Clp1 is a Cdc14-family phosphatase that reverses mitotic Cdk1 phosphorylation. Despite evolutionary conservation, Clp1’s mammalian orthologs do not share this function. Rather, higher eukaryotic Cdc14 enzymes act in DNA repair, ciliogenesis, and gene regulation. To examine if Clp1 regulates gene expression, we compared the transcriptional profiles of cells lacking Clp1 function to that of wildtype. Because clp1∆ cells are sensitive to the actin depolymerizing drug, LatrunculinA, we also investigated whether a transcriptional response was involved. Our results indicate that Clp1 does not detectably affect gene expression and highlight the organism-specific functions of this conserved phosphatase family.

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Project description:The clp1 gene is a b-regulated gene that is required for clamp formation during the biotrophic growth phase of U. maydis. Interaction studies demonstrated that Clp1 imnteracts with Rbf1, the central regulator of pathogenciity. To exclude b-dependent effects the experiments were performed in a b-null background to analyze clp1-depndent effects on rbf controlled gene regulation Strains UKH156 (nitrate inducible Rbf1 expression) and UKH164 (arabinose-inducible Clp1 and and nitrate-inducible Rbf1 expression) were grown for 12h in liquid array medium/nitrate/arabinose.

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