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The spliceosome factor sart3 regulates hematopoietic stem/progenitor cell development in zebrafish through the p53 pathway

ABSTRACT: Hematopoietic stem cells (HSCs) possess the potential for self-renew and the capacity, throughout life, to differentiate into all blood cell lineages. Yet, the mechanistic basis for HSC development remains largely unknown. In this study, we characterized a zebrafish smu471 mutant with hematopoietic stem/progenitor cell (HSPC) defects and found that sart3 was the causative gene. RNA expression profiling of the sart3smu471 mutant revealed spliceosome and p53 signaling pathway to be the most significantly enriched pathways in the sart3smu471 mutant. Knock down of p53 rescued HSPC development in the sart3smu471 mutant. Interestingly, the p53 inhibitor, mdm4, had undergone an alternative splicing event in the mutant. Restoration of mdm4 partially rescued HSPC deficiency. Thus, our data suggest that HSPC proliferation and maintenance require sart3 to ensure the correct splicing and expression of mdm4, so that the p53 pathway is properly inhibited to prevent definitive hematopoiesis failure. This study expands our knowledge of the regulatory mechanisms that impact HSPC development and sheds light on the mechanistic basis and potential therapeutic use of sart3 in spliceosome-mdm4-p53 related disorders.

ORGANISM(S): Danio rerio

PROVIDER: GSE182560 | GEO | 2021/09/08

REPOSITORIES: GEO

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Project description:Purpose: Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for craniofacial and rib defects in patients with Cerebrocostomandibular Syndrome (CCMS). However, why the mutations in SNRPB causes tissue specific CCMS abnormalities such as craniofacial defects are unknown. We hypothesize that splicing of tissue specific transcripts required for craniofacial development is disrupted due to SNRPB deficiency, during embryonic development. The purpose of the study was to identify those developmentally important transcripts that are dysregulated due to Snrpb mutation. Methods: We mated Snrpbloxp mice that we have developed with commercially available Wnt-1-Cre2 transgenic mice to remove one allele of Snrpb from neural crest cells. The mutant (Snrpb \ncc+/-) embryos showed craniofacial abnormalities at E9.5 and onward. We collected morphologically normal embryos at E9.0 for both Snrpb wildtype and Snrpbncc+/- mutants. We pooled two embryo heads of similar somites for each genotype and extracted the RNA for RNAseq. Results: We found that Snrpbncc+/- mutants show aberrant splicing that includes both increased exon skipping and intron retention. A strong tendency towards increased exon skipping and intron inclusion in the mutant samples were observed; there were more SE (273 in Het versus 83 in WT) and RI (191 in Het versus 21 in WT). We identified 13 transcripts required for craniofacial development or stem cell development with significant increases in exon skipping including Smad2, Rere and Pou2f1. From pathway analysis of differentially expressed genes, we found they were associated with the spliceosome and the P53-pathway. We also found an increase in splicing of two P53 regulator, Mdm2 and Mdm4. Conclusions: We propose that abnormal splicing underlies the defects found in Snrpbncc+/-mutant embryos. We confirmed differential splicing of the P53 regulators, Mdm2 and Mdm4 and identified several transcripts important for craniofacial development which were abnormally spliced in Snrpbncc+/- embryos.

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The spliceosome factor sart3 regulates hematopoietic stem/progenitor cell development in zebrafish through the p53 pathway

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