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Spliceosomal protein eftud2 mutation leads to p53-dependent apoptosis in zebrafish neural progenitors.

ABSTRACT: Haploinsufficiency of EFTUD2 (Elongation Factor Tu GTP Binding Domain Containing 2) is linked to human mandibulofacial dysostosis, Guion-Almeida type (MFDGA), but the underlying cellular and molecular mechanisms remain to be addressed. We report here the isolation, cloning and functional analysis of the mutated eftud2 (snu114) in a novel neuronal mutant fn10a in zebrafish. This mutant displayed abnormal brain development with evident neuronal apoptosis while the development of other organs appeared less affected. Positional cloning revealed a nonsense mutation such that the mutant eftud2 mRNA encoded a truncated Eftud2 protein and was subjected to nonsense-mediated decay. Disruption of eftud2 led to increased apoptosis and mitosis of neural progenitors while it had little effect on differentiated neurons. Further RNA-seq and functional analyses revealed a transcriptome-wide RNA splicing deficiency and a large amount of intron-retaining and exon-skipping transcripts, which resulted in inadequate nonsense-mediated RNA decay and activation of the p53 pathway in fn10a mutants. Therefore, our study has established that eftud2 functions in RNA splicing during neural development and provides a suitable zebrafish model for studying the molecular pathology of the neurological disease MFDGA.

SUBMITTER: Lei L

PROVIDER: S-EPMC5389467 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Spliceosomal protein eftud2 mutation leads to p53-dependent apoptosis in zebrafish neural progenitors.

Lei Lei L Yan Shou-Yu SY Yang Ran R Chen Jia-Yu JY Li Yumei Y Bu Ye Y Chang Nannan N Zhou Qinchao Q Zhu Xiaojun X Li Chuan-Yun CY Xiong Jing-Wei JW

Nucleic acids research 20170401 6

Haploinsufficiency of EFTUD2 (Elongation Factor Tu GTP Binding Domain Containing 2) is linked to human mandibulofacial dysostosis, Guion-Almeida type (MFDGA), but the underlying cellular and molecular mechanisms remain to be addressed. We report here the isolation, cloning and functional analysis of the mutated eftud2 (snu114) in a novel neuronal mutant fn10a in zebrafish. This mutant displayed abnormal brain development with evident neuronal apoptosis while the development of other organs appea ...[more]

PMID: 27899647

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Project description:The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated.Mammalian cells that are SMC3 deficient fail to expand in a clonal population. In order to shed light on the underlying mechanism, experiments were conducted in zebrafish embryos in which cell competence to undergo apoptosis is acquired at specific stages of development and affects tissue morphogenesis. Zebrafish Smc3 is 95% identical to the human protein, is maternally contributed, and is expressed ubiquitously at all developmental stages. Antisense-mediated loss of Smc3 function leads to increased apoptosis in Smc3 expressing cells of the developing tail and notocord causing morphological malformations. The apoptosis and the ensuing phenotype can be suppressed by injection of a p53-specific MO that blocks the generation of endogenous p53 protein. Results in human cells constitutively lacking p53 or BAX, confirmed that a p53-dependent pathway mediates apoptosis in SMC3-deficient cells. A population of aneuploid cells accumulated in zebrafish embryos following Smc3-knockdown whereas in human cells the transient downregulation of SMC3 level lead to the generation of cells with amplified centrosome number.Smc3 is required for normal embryonic development. Its deficiency affects the morphogenesis of tissues with high mitotic index by triggering an apoptotic cascade involving p53 and the downstream p53 target gene bax. Cells with low SMC3 level display centrosome abnormalities that can lead to or are the consequence of dysfunctional mitosis and/or aneuploidy. Collectively the data support the view that SMC3 deficiency affects chromosomal stability leading to the activation of p53-dependent mitotic checkpoint.

Project description:DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the ?-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.

Dataset Information

Spliceosomal protein eftud2 mutation leads to p53-dependent apoptosis in zebrafish neural progenitors.

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Spliceosomal protein eftud2 mutation leads to p53-dependent apoptosis in zebrafish neural progenitors.

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