PPP6R3-mediated dephosphorylation regulates mRNA translation during spermatogonial differentiation
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ABSTRACT:
PROVIDER: PRJNA1165730 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30811975_1.fastq.gz | Fastqsanger.gz | |||
| SRR30811975_2.fastq.gz | Fastqsanger.gz | |||
| SRR30811976_1.fastq.gz | Fastqsanger.gz | |||
| SRR30811976_2.fastq.gz | Fastqsanger.gz | |||
| SRR30811977_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein dephosphorylation mediated by phosphatases regulates spermatogenesis. However, which proteins are dephosphorylated and how they regulate spermatogenesis are largely unknown. Here, we show that germline-specific deletion of protein phosphatase 6 regulatory subunit 3 (PPP6R3), which determines substrate specificity of protein phosphatase 6 catalytic subunit, causes abnormal spermatogonial differentiation and male infertility, accompanied by translation inhibition. PPP6R3 directly interacts with EIF3C and EIF4G1 in KIT+ spermatogonia. Decreased levels of non-phosphorylated EIF3C and EIF4G1 after PPP6R3 deletion attenuate their enrichment for mRNAs associated with spermatogonial differentiation, and increased phosphorylation levels promote their degradation. Specifically, the phosphorylation status both of EIF3CS39 and EIF4G1S1217 are significantly upregulated in mutant mice. Overexpression of EIF3CS39A and EIF4G1S1217A mutants in Ppp6r3-cKO spermatogonial progenitor cells compensates for their differentiation efficiency by upregulating the translation rates of differentiation-associated mRNAs. Our findings demonstrate that dephosphorylation of EIF3C and EIF4G1 mediated by PPP6R3 is critical for translation activation during spermatogonial differentiation.
2025-06-20 | GSE278121 | GEO
Project description:In the testis, a subset of spermatogonia retains stem cell potential, while others differentiate to eventually become spermatozoa. This delicate balance must be maintained, as defects can result in testicular cancer or infertility. Currently, little is known about the gene products and signaling pathways directing these critical cell fate decisions. Retinoic acid (RA) is a requisite driver of spermatogonial differentiation and entry into meiosis, yet the mechanisms activated downstream are undefined. Here, we determined a requirement for RA in the expression of KIT, a receptor tyrosine kinase essential for spermatogonial differentiation. We found that RA signaling utilized the PI3K/AKT/mTOR signaling pathway to induce the efficient translation of mRNAs for Kit, which are present but not translated in undifferentiated spermatogonia. Our findings provide an important molecular link between a morphogen (RA) and the expression of KIT protein, which together direct the differentiation of spermatogonia throughout the male reproductive lifespan.
| S-EPMC4268412 | biostudies-literature
Project description:Spermatogonial differentiation is a developmental process that is essential for spermatogenesis, but the molecular and cellular changes that germ cells must undergo to transition from undifferentiated spermatogonia to differentiating spermatogonia remain largely undefined. Retinoic acid (RA) is necessary and sufficient for spermatogonial differentiation. Using the postnatal mouse testis, we examine the transcriptome changes that accompany spermatogonial differentiation. Spermatogenesis was synchronized by administration of potent and selective RA synthesis inhibitor; as a result, testes contained only undifferentiated spermatogonia. Then, the inhibitor was discontinued, and mice were given a single dose of exogenous RA to initiate spermatogonial differentiation. We measured transcriptomes in FACS-enriched germ cells either before RA administration, when the cells correspond to Aal spermatogonia (and a minor contribution of spermatogonial stem cells) or at two points after RA administration, when the cells correspond to A1 or A3 differentiating spermatogonia. The results of this study reveal the full transcriptome changes accompanying spermatogonial differentiation in the mouse.
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Project description:A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference of bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid-induced differentiation. To accommodate this elevated energy demand, retinoic acid signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. In addition, inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway blocked the formation of c-Kit+ differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon retinoic acid-induced differentiation by both RNA-seq analyses and quantitative proteomics. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration which is required for spermatogonial proliferation and differentiation.
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Project description:Increasing evidence suggests that Long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of novel intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding-protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well-conserved between human and mouse, its locus, gene structure and function is preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. In order to perform an unbiased search for downstream signaling pathways perturbed by LncMyodD downregulation, microarrays were performed on myoblasts treated with control vs LncMyoD shRNAs. Total RNA was extracted using the TRIzol reagent (Invitrogen) and purified with Qiagen RNeasy separation columns (Qiagen) from myoblasts treated with control vs. LncMyoD shRNA. First-strand cDNA was synthesized and hybridized to GeneChip Mouse Genome 430 2.0 Array (Affymetrix).
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Project description:METTL3 catalyzes the formation of N6-methyl-adenosine (m6A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m6A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m6A modification in spermatogenesis and reproduction in mammals.
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Project description:Translation is a fundamental biological process, which defines every aspect of cell physiology. Dysregulation of protein synthesis is associated with the diseases, such as ribosomopathies, diabetes, and cancer. To examine translation dysregulation in vivo, we employed RNA interference to knockdown m-subunit of the translation initiation factor eIF3 exclusively in mouse liver. We observed an increase in the number of monosomes and multiple signs of impaired translation. We further characterized the early cellular response using transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses. The major response of hepatocytes to eIF3m deficiency was associated with the changes in mRNA expression, with almost no effect on translation efficiency for particular mRNAs. The transcription changes fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins, dephosphorylation of eIF2α, and inhibition of rRNA processing) and cell metabolism (lipid, amino acid, nucleic acid, and drug metabolism). Overall, this work uncovers a new mode of regulation of protein synthesis mediated by the inhibition of translation initiation. It also highlights advantages of RNAi-based in vivo approach for studying regulatory network associated with essential factors involved in mammalian translation.
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Project description:We demonstrate that Protein Phosphatase PfPPM2 regulates both asexual and sexual development of human malaria parasite P. falciparum. It is involved in the division of the parasite during asexual development and promotes its sexual differentiation. Phosphoproteomics studies revealed that PfPPM2 targets in the parasite include key regulators of protein translation and chromatin remodelling. We demonstrate that it regulates dephosphorylation of S33 of Heterochromatin Protein 1 (HP1), a regulator of heritable gene silencing and contributes to both mitotic proliferation as well as sexual differentiation of the parasite into sexual forms. Detailed investigations revealed that PfPPM2 mediated dephosphorylation of HP1 at S33 is a signal for parasite sexual conversion. HP1- S33 dephosphorylation impairs its interaction with trimethylated form of histone 3 (H3) at lysine 9 (H3K9me3), which is known to stabilize the heterochromatin. As a result, the expression of transcription of ap2g-which is repressed by HP1-H3K9me3-was stimulated resulting in sexual conversion of the parasite. Interestingly, the state of HP1-S33 phosphorylation seems to largely reflect the fate of the parasite-its optimal phosphorylation is important for asexual division whereas it is dephosphorylated form favours sexual differentiation. PfPPM2 also regulates protein synthesis in the parasite by repressing a signaling pathway involved in the phosphorylation of initiation factor eIF2α, which are likely to contribute to parasite division and possibly differentiation.
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Project description:In vitro and in vivo aging of mouse spermatogonial stem cells alters stem cell function based on quantitative spermatogonial stem cell transplantation analyses. We used microarrays to identify differential gene expression in vitro and in vivo aged spermatogonial stem cells to identify potential causes of observed phenotypic differences in aged spermatogonial stem cell function. Spermatogonial stem cells were isolated from young and serial-transplanted aged mouse donors and cultured for short and long periods. Spermatogonial stem cells were isolated from cultures and subjected to microarray analysis to identify differential gene expression.
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2023-03-11 | PXD032277 | Pride