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Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA splicing in pontocerebellar hypoplasia

ABSTRACT: Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA splicing in pontocerebellar hypoplasia

PROVIDER: PRJNA635177 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11852087.fastq.gz	Fastqsanger.gz
	SRR11852088.fastq.gz	Fastqsanger.gz
	SRR11852089.fastq.gz	Fastqsanger.gz
	SRR11852090.fastq.gz	Fastqsanger.gz
	SRR11852091.fastq.gz	Fastqsanger.gz

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Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA splicing in pontocerebellar hypoplasia

Project description:Our work defines the molecular principles of intron excision in humans and provides evidence that destabilized TSEN contributes to a PCH phenotype by impairing tRNA splicing at the intron-excision step.

2021-06-18 | GSE151236 | GEO

The Clp1 R140H mutation alters tRNA metabolism and mRNA 3’ processing in mouse models of pontocerebellar hypoplasia

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.

2021-09-20 | GSE176183 | GEO

Molecular Characterization of Pontocerebellar Hypoplasia

Project description:Molecular Characterization of Pontocerebellar Hypoplasia

| PRJNA1062911 | ENA

Molecular Characterization of Pontocerebellar Hypoplasia

Project description:Molecular Characterization of Pontocerebellar Hypoplasia

| PRJNA1062912 | ENA

Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia.

Project description:Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

| S-EPMC8479045 | biostudies-literature

Autosomal-Recessive Mutations in the tRNA Splicing Endonuclease Subunit TSEN15 Cause Pontocerebellar Hypoplasia and Progressive Microcephaly.

Project description:The tRNA splicing endonuclease is a highly evolutionarily conserved protein complex, involved in the cleavage of intron-containing tRNAs. In human it consists of the catalytic subunits TSEN2 and TSEN34, as well as the non-catalytic TSEN54 and TSEN15. Recessive mutations in the corresponding genes of the first three are known to cause pontocerebellar hypoplasia (PCH) types 2A-C, 4, and 5. Here, we report three homozygous TSEN15 variants that cause a milder version of PCH2. The affected individuals showed progressive microcephaly, delayed developmental milestones, intellectual disability, and, in two out of four cases, epilepsy. None, however, displayed the central visual failure seen in PCH case subjects where other subunits of the TSEN are mutated, and only one was affected by the extensive motor defects that are typical in other forms of PCH2. The three amino acid substitutions impacted the protein level of TSEN15 and the stoichiometry of the interacting subunits in different ways, but all resulted in an almost complete loss of in vitro tRNA cleavage activity. Taken together, our results demonstrate that mutations in any known subunit of the TSEN complex can cause PCH and progressive microcephaly, emphasizing the importance of its function during brain development.

| S-EPMC5005448 | biostudies-literature

A homozygote variant in the tRNA splicing endonuclease subunit 54 causes pontocerebellar hypoplasia in a consanguineous Iranian family.

Project description:BackgroundHomozygous loss-of-function mutations in TSEN54 (tRNA splicing endonuclease subunit 54; OMIM: 608755) cause different types of pontocerebellar hypoplasias (PCH) including PCH2, PCH4, and PCH5. The study aimed to determine the possible genetic factors contributing to PCH phenotypes in two affected male infants in an Iranian family.MethodsWe subjected two affected individuals in a consanguineous Iranian family. To systematically investigate the susceptible gene(s), whole-exome sequencing was performed on the proband and a novel identified variant was confirmed by Sanger sequencing. We also analyzed 26 relatives in three generations using PCR-restriction fragment length polymorphism (PCR-RFLP) followed and confirmed by Sanger sequencing.ResultsPhysical and medical examinations confirmed PCH in the patients. Besides, the proband showed bilateral moderate sensorineural hearing loss and structural heart defects as the novel phenotypes. The molecular findings also verified that two affected individuals were homozygote for the novel synonymous variant, NM_207346.2: c.1170G>A; p.(Val390Val), in TSEN54. PCR-RFLP and Sanger sequencing elucidated that the parents and 16 relatives were heterozygote for the novel variant.ConclusionWe identified a novel synonymous variant, c.1170G>A, in TSEN54 associated with PCH in an Iranian family. Based on this study, we strongly suggest using "TSENopathies" to show the overlapped phenotypes among different types of PCH resulted from TSEN causative mutations.

| S-EPMC7549571 | biostudies-literature

The Clp1 R140H mutation alters tRNA metabolism and mRNA 3’ processing in mouse models of pontocerebellar hypoplasia

Project description:The Clp1 R140H mutation alters tRNA metabolism and mRNA 3’ processing in mouse models of pontocerebellar hypoplasia

| PRJNA735232 | ENA

Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Project description:To gain functional insights into human TSEN/CLP1, we designed an expression vector series based on the MultiBac system that allows combinatorial protein complex production in insect and mammalian cells by utilizing a CMV/p10 dual promoter. Using this system, we were able to assemble and purify functional heterotetrameric TSEN and a heteropentameric complex including the RNA kinase CLP1 from infected insect cells. Individual proteins of the complexes were identified using in-gel digestion and LC-MS/MS analysis. To gain atomistic insights into the molecular architecture of the human TSEN complex, we set out to characterize the TSEN15–34 heterodimer by X-ray crystallography. Despite extensive crystallization trials, full-length TSEN15–34 did not yield any crystals. To define a crystallizable core complex, we subjected the full-length complex to limited proteolysis with subsequent size exclusion chromatography. Primarily, we observed two comigrating polypeptide species. The two polypeptide species were identified using in-gel digestion followed by LC-MS/MS analysis. This led to the identification of the polypeptide species corresponding to residues 23 to 170 of TSEN15 and residues 208 to 310 of TSEN34 covering the predicted conserved nuclease domains.

2021-06-17 | PXD019034 | Pride

Recessive mutation in EXOSC3 associates with mitochondrial dysfunction and pontocerebellar hypoplasia

Project description:Recessive mutations in EXOSC3, encoding a subunit of the human RNA exosome complex, cause Pontocerebellar hypoplasia type 1b (PCH1B). We report a boy with severe muscular hypotonia, psychomotor retardation, progressive microcephaly, and cerebellar atrophy. Biochemical abnormalities comprised mitochondrial Complex I and PDHc deficiency. Whole exome sequencing uncovered a known EXOSC3-mutation p.(D132A) as the underlying cause. In patient fibroblasts, >50% of the EXOSC3 protein was trapped in the cytosol. mtDNA-copy numbers in muscle were reduced to 40%, but mutations in the mtDNA and nuclear mitochondrial genes were excluded. RNA-seq of patient muscle showed highly increased mRNA-copy numbers, especially for genes encoding structural subunits of OXPHOS-complexes I, III, and IV, possibly due to reduced degradation by a dysfunctional exosome complex. This is the first case of mitochondrial dysfunction associated with an EXOSC3 mutation, which expands the phenotypic spectrum of PCH1B. We discuss the links between exosome and mitochondrial dysfunction.

2017-08-22 | GSE99715 | GEO

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