Impairment of the tRNA-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia

AbstractIntrons 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.

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

Nature Communications ◽

10.1038/s41467-021-25870-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Samoil Sekulovski ◽

Pascal Devant ◽

Silvia Panizza ◽

Tasos Gogakos ◽

Anda Pitiriciu ◽

...

Keyword(s):

Transfer Rna ◽

Endonuclease Activity ◽

Pontocerebellar Hypoplasia ◽

Trna Processing ◽

X Ray ◽

Trna Splicing ◽

Cleavage Activity ◽

Differential Scanning Fluorimetry ◽

Mature Domain ◽

Site Recognition

AbstractIntrons 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.

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

The American Journal of Human Genetics ◽

10.1016/j.ajhg.2016.08.009 ◽

2016 ◽

Vol 99 (3) ◽

pp. 785 ◽

Cited By ~ 8

Author(s):

Martin W. Breuss ◽

Tipu Sultan ◽

Kiely N. James ◽

Rasim O. Rosti ◽

Eric Scott ◽

...

Keyword(s):

Autosomal Recessive ◽

Pontocerebellar Hypoplasia ◽

Trna Splicing ◽

Recessive Mutations

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

Molecular Genetics & Genomic Medicine ◽

10.1002/mgg3.1413 ◽

2020 ◽

Vol 8 (10) ◽

Author(s):

Afrooz Sepahvand ◽

Ehsan Razmara ◽

Fatemeh Bitarafan ◽

Mohammad Galehdari ◽

Ali Reza Tavasoli ◽

...

Keyword(s):

Pontocerebellar Hypoplasia ◽

Trna Splicing ◽

Iranian Family

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2110730118 ◽

2021 ◽

Vol 118 (39) ◽

pp. e2110730118

Author(s):

Caitlin E. Monaghan ◽

Scott I. Adamson ◽

Mridu Kapur ◽

Jeffrey H. Chuang ◽

Susan L. Ackerman

Keyword(s):

Mouse Models ◽

Motor Neurons ◽

Messenger Rna ◽

Cerebellar Nuclei ◽

Trna Genes ◽

Homozygous Mutation ◽

Pontocerebellar Hypoplasia ◽

Trna Splicing ◽

Mrna Cleavage ◽

A Site

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 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 were also 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 impaired mRNA 3′ processing may contribute to pathogenesis in PCH10.

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Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

10.1101/2021.07.09.451847 ◽

2021 ◽

Author(s):

Casey A Schmidt ◽

Lucy Y Min ◽

Michelle H McVay ◽

Joseph D Giusto ◽

John C Brown ◽

...

Keyword(s):

Rna Processing ◽

Brain Size ◽

Neurological Diseases ◽

Premature Death ◽

Pontocerebellar Hypoplasia ◽

Trna Splicing ◽

Intervening Sequences ◽

Brain Lobe ◽

Processing Factors

Mature tRNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, we characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, we also found increased cell death in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that we can successfully model PCH in a genetically-tractable invertebrate.

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