Mutations in <i>Drosophila</i> tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

Ca, Schmidt; Ly, Min; Mh, McVay; Jd, Giusto; Jc, Brown; Hr, Salzler; Matera, A. Gregory

doi:10.1101/2021.07.09.451847

Cited by 4 publications

(11 citation statements)

References 41 publications

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“…Previous studies on human samples and animal models indicated that hypoplasia results from degeneration and cell death, respectively (Barth et al, 2007; Kasher et al, 2011; Schmidt et al, 2022). We therefore investigated whether elevated levels of apoptosis could explain the reduced size of PCH2a cerebellar and neocortical organoids.…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, in different systems, tRNAs can directly regulate apoptosis (Avcilar-Kucukgoze and Kashina, 2020; Mei et al, 2010). Neurodegeneration and apoptosis have been suggested to occur in the cerebellum of affected individuals with PCH based on neuropathological observations (Barth et al, 2007) and in animal models of PCH (Ishimura et al, 2014; Kasher et al, 2011; Schmidt et al, 2022), respectively. However, our analysis of cCas3-positive cells did not reveal any differences in apoptosis between PCH2a and control cerebellar and neocortical organoids at D30 and D50.…”

Section: Discussionmentioning

confidence: 99%

“…Of the frequently used model organisms, only mouse and chicken share the amino acid residue (Budde et al, 2008). A recently developed fly model of PCH shows defects in brain development, death at larval stages, and apoptosis upon loss of function of the TSEN54 ortholog, but the relevance to the brain region-specific clinical phenotype is unclear (Schmidt et al, 2022). In zebrafish, loss of tsen54 function leads to cell death in the brain during development (Kasher et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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Human organoid model of PCH2a recapitulates brain region-specific pathology

Kagermeier

Hauser

Sarieva

et al. 2022

Preprint

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Pontocerebellar hypoplasia type 2 a (PCH2a) is a rare, autosomal recessive neurogenetic disorder. Affected individuals present with early and severe neurological impairment. The anatomical hallmark of PCH2a is the hypoplasia of the cerebellum and pons accompanied by progressive microcephaly over the first years of life (OMIM #277470). Treatment options are limited and symptomatic. PCH2a results from a homozygous founder variant in the TSEN54 gene (OMIM *608755), which encodes a tRNA splicing endonuclease complex subunit. A recent study revealed altered tRNA pools in fibroblasts from affected individuals with PCH2a. However, the pathological mechanism underlying the hypoplasia of the cerebellum and the progressive microcephaly is unknown due to a lack of a model system. Leveraging recent progress in organoid generation, we developed human models of PCH2a using brain region-specific organoids. We, therefore, obtained skin biopsies from three affected males with genetically confirmed PCH2a and derived induced pluripotent stem cells (iPSCs). Cerebellar and neocortical organoids were differentiated from control and affected iPSCs and showed expression of TSEN54. In line with neuroimaging findings in affected individuals, PCH2a cerebellar organoids are reduced in size compared to controls starting early in differentiation. While neocortical PCH2a organoids also show growth deficits, they are less pronounced than those in cerebellar organoids, reminiscent of the progressive microcephaly in PCH2a. In addition, we do not find evidence of increased apoptosis in PCH2a organoids compared to controls in contrast to what has been suggested in loss-of-function animal models. We have generated a human model of PCH2a, which will allow to decipher disease mechanisms in depth in order to explain how a variant in a ubiquitously expressed gene involved in tRNA metabolism causes pathology in specific brain regions.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Human organoid model of PCH2a recapitulates brain region-specific pathology

Kagermeier

Hauser

Sarieva

et al. 2022

Preprint

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“…A rare disease caused by a shared mechanism or a family of unrelated, yet similar diseases PCH is a rare autosomal recessive disease primarily caused by mutations in RNA processing factors. In addition to the many fly model organisms mentioned above, several new fly PCH models have recently been developed (Morton et al, 2020;Schmidt et al, 2021;Slavotinek et al, 2020), which may be useful tools to better characterize and understand PCH. PCH appears to primarily be caused by dysfunction in RNA processing machinery, with a couple of exceptions.…”

Section: Pchmentioning

confidence: 99%

Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex

2022

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The splicing of transfer RNA (tRNA) introns is a critical step of tRNA maturation, for intron-containing tRNAs. In eukaryotes, tRNA splicing is a multi-step process that relies on several RNA processing enzymes to facilitate intron removal and exon ligation. Splicing is initiated by the tRNA splicing endonuclease (TSEN) complex which catalyzes the excision of the intron through its two nuclease subunits. Mutations in all four subunits of the TSEN complex are linked to a family of neurodegenerative and neurodevelopmental diseases known as pontocerebellar hypoplasia (PCH). Recent studies provide molecular insights into the structure, function, and regulation of the eukaryotic TSEN complex and are beginning to illuminate how mutations in the TSEN complex lead to neurodegenerative disease. Using new advancements in the prediction of protein structure, we created a three-dimensional model of the human TSEN complex. We review functions of the TSEN complex beyond tRNA splicing by highlighting recently identified substrates of the eukaryotic TSEN complex and discuss mechanisms for the regulation of tRNA splicing, by enzymes that modify cleaved tRNA exons and introns. Finally, we review recent biochemical and animal models that have worked to address the mechanisms that drive PCH and synthesize these studies with previous studies to try to better understand PCH pathogenesis.

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“…Mammalian TSEN additionally associates with the RNA kinase CLP1 by uncharacterized interactions 1,30 . While in vitro experiments showed that CLP1 is dispensable for pre-tRNA splicing 10,19 , in vivo and in-cell studies connected mutations in CLP1 to impaired pre-tRNA splicing and neurodegeneration 7,8,[31][32][33] . Indeed, mutations in CLP1 and all four subunits of human TSEN have been associated with pontocerebellar hypoplasia (PCH), a broad group of inherited neurodegenerative disorders [2][3][4][5][6][7][8]31,34 .…”

Section: Mainmentioning

confidence: 99%

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

Sekulovski

Sušac

Stelzl

et al. 2022

Preprint

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The heterotetrameric human transfer RNA (tRNA) splicing endonuclease (TSEN) catalyzes the excision of intronic sequences from precursor tRNAs (pre-tRNAs). Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH). The three-dimensional (3D) assembly of TSEN/CLP1, the mechanism of substrate recognition, and the molecular details of PCH-associated mutations are not fully understood. Here, we present cryo-electron microscopy structures of human TSEN with intron-containing pre-tRNATyrGTA and pre-tRNAArgTCT. TSEN exhibits broad structural homology to archaeal endonucleases9 but has evolved additional regulatory elements that are involved in handling and positioning substrate RNA. Essential catalytic residues of subunit TSEN34 are organized for the 3' splice site which emerges from a bulge-helix configuration. The triple-nucleotide bulge at the intron/3'-exon boundary is stabilized by an arginine tweezer motif of TSEN2 and an interaction with the proximal minor groove of the helix. TSEN34 and TSEN54 define the 3' splice site by holding the tRNA body in place. TSEN54 adapts a bipartite fold with a flexible central region required for CLP1 binding. PCH-associated mutations are located far from pre-tRNA binding interfaces explaining their negative impact on structural integrity of TSEN without abrogating its catalytic activity in vitro10. Our work defines the molecular framework of pre-tRNA recognition and cleavage by TSEN and provides a structural basis to better understand PCH in the future.

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

Cited by 4 publications

References 41 publications

Human organoid model of PCH2a recapitulates brain region-specific pathology

Human organoid model of PCH2a recapitulates brain region-specific pathology

Recent insights into the structure, function, and regulation of the eukaryotic transfer RNA splicing endonuclease complex

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

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