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Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing

ABSTRACT: Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here we show that knockout (KO) of Dars2 significantly impairs the maintenance of HSCs and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3 labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathway in HSPCs. Our study reveals that Dars2 plays an unprecedented role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Bone Marrow

SUBMITTER: Kailing Li

LAB HEAD: Bin Guo

PROVIDER: PXD045562 | Pride | 2023-09-26

REPOSITORIES: Pride

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Project description:Microarray experiment on each time point and genotype was performed in technical duplicates (ie. a single RNA preparation of pooled kidneys of one genotype used for two separate target preparations). Data from a total of 8 independent arrays were used in this study, 2 arrays were used for RNA samples from E18.5 control kidneys, 2 for E18.5 Lim1 conditional mutant kidneys, 2 for E14.5 control kidneys, and the other 2 for E14.5 Lim1 conditional mutant kidneys. Data generated from all arrays that satisfied the preliminary analysis were exported and loaded into DNA-Chip Analyzer (dChip2004), where statistical and comparative analyses were performed to verify the data. The data were normalized using the default normalization method. Briefly, an iterative procedure was used to identify an invariant set of probes, which presumably consisted of non-differentially expressed genes. A piecewise-linear running median curve was then calculated and used as the normalization curve. After normalization, all arrays had similar brightness. Median intensities around 155 (between 155 to 158) were obtained after normalization. Percent gene present (P call%) values between 55.7% and 63.6% were observed using default detection p-value cut offs (a1=0.04 and a2=0.06). Array outlier (%) and single outlier (%) were detected at ranges from 0.016% to 0.080% and from 0.009% to 0.045%. Expression data obtained from all arrays used are provided. Normalized data were exported in a tab delimited text format. Fold changes of each transcript from different samples were calculated and sorted using Microsoft Excel 5.0 software. Signal obtained from control kidney samples were used as an experiment to compare to the signal obtained from Lim1 conditional mutant kidneys that was designated as a baseline. A 2-fold change in the means of signal obtained from experimental duplicates and those from baseline duplicates was used as the criterion to identify differentially expressed transcripts. To ensure the quality of the data, probe sets that showed a fold change between duplicates greater than between the experimental mean and baseline mean were removed. To study only genes that showed consistent expression on experimental chips, probe sets that did not show consistent present calls in the experimental duplicates were removed. To focus on genes with a significant fold change between the experiment and the baseline, only probe sets that the product of their experimental mean and fold change were more than 100 were retained. To produce a compact differentially expressed gene list, the probe set list was sorted within Microsoft Excel based on Locus Link number and redundant entries were removed. Our experimental design description and the data format provided in the Additional files fulfill the MIAME (minimum information about a microarray experiment) standards.

Project description:Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration. We developed hepatocyte-specific Socs3 knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after 2/3 partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after 2/3 PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic kinases extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor (EGF) or interleukin 6 (IL-6) stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes after PH, which fall into diverse physiologic categories. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma (HCC) at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiologic and neoplastic proliferative processes in the liver, and may act as a tumor suppressor. Experiment Overall Design: Hepatocyte-specific excision of the Socs3 gene was achieved by breeding Socs3 fl/fl mice with mice expressing the Cre recombinase transgene under control of the albumin promoter (Alb-Cre+), yielding Socs3 h-KO mice. Socs3 fl/fl, Alb-Cre- littermates were used as controls for all experiments, and are henceforth referred to as littermates. All mice (C57BL/6) were free of Helicobacter species, housed in a specific pathogen free facility with 12-h light/dark cycles with free access to standard food and water. 2/3 PH and sham operations were performed as previously described (15, 50) (n=3-6 mice per genotype per time point). Liver remnants were weighed after removal of necrotic stumps and sutures, and compared to post-operative body weight. For HCC experiments, a single i.p. injection of DEN (5mg/kg, Sigma) was performed 12-14 d after birth. For short time points, a single injection of DEN (100mg/kg) (31) was given to 4 wk old mice. At indicated time points, mice were sacrificed by CO2 inhalation. All animal studies were carried out under approved IACUC protocols at the University of Washington.

			Action	DRS
	Dars2-KO1.raw	Raw
	Dars2-KO2.raw	Raw
	Dars2-KO3.raw	Raw
	WT1.raw	Raw
	WT2.raw	Raw

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Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing

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