image: NAT10 in post-transcriptionally regulating DMRT1 and CCNA2 substrates essential for the maintenance of spermatogonial homeostasis.
Credit: ©Science China Press
This study is led by Professor Jianqiang Bao's research team from Center for Reproduction and Genetics, the First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, in collaboration with the team led by Director Shen Zhang from the Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-XIANGYA，and Professor Yunfang Zhang from Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai.
The study employed in-house-generated, highly efficient, tamoxifen-inducible Ddx4-driven Nat10 knockout (KO) and Nat10-ScKO mouse model, created by crossing Stra8-Cre mice with Nat10lox/lox mice. The result showed that NAT10 is required to maintain spermatogonial proliferation and differentiation in both pubertal and adult mouse testes.
The study found that Nat10 knockout in both mouse models resulted in infertility with significantly reduced testicular sizes. Histological analysis using hematoxylin and eosin (H&E) staining revealed a prominent loss of germ cells upon Nat10 KO and by P12, most germ cells were depleted in Nat10-ScKO testes, as evidenced by promyelocytic leukaemia zinc finger protein (PLZF) and germ cell nuclear acidic peptidase (GCNA) staining. Immunoblotting using antibodies against PLZF, mouse vasa homologue (MVH), and C-kit further confirmed that the significant reduction in both undifferentiated (GFRα1+ and PLZF+) and differentiated (c-Kit+) spermatogonia were significantly reduced in Nat10-DcKO testes at P7.
These findings suggest that NAT10 is essential for spermatogonial proliferation and differentiation. Next, they investigated whether Nat10 is necessary for the maintenance of the steady-state spermatogonial pool in adult mouse testes. Results show that prior to 18 days post tamoxifen (dpt), no discernible morphological differences between wild-type (WT) and Nat10-DcKO testes were not discernible by H&E staining. Nonetheless, most germ cells were depleted from the seminiferous tubules in Nat10-DcKO testes, leading to the reduced testicular weight, as accompanied by frequent vacuole formation starting at 32 dpt and the resultant “Sertoli cell-only syndrome”. These data indicate that Nat10 is essential for the maintenance of steady-state homeostasis of spermatogonia in adult testes.
Having validated the indispensable roles of Nat10 for both prepubertal spermatogonial development and adult spermatogonial maintenance, they next elucidated the molecular transcriptome impacted in Nat10-null spermatogonia. Gene Ontology (GO) analysis suggested that Nat10-down-regulated genes are mostly relevant to cell cycle and cell division as well as meiotic cell cycle, compared with Nat10-up-regulated genes. Moreover, the expression levels of some key genes involved in spermatogonial maintenance mostly decreased. Nat10 is thus far the only known ‘writer’ for the ac4C mark on RNA substrates, they detect ac4C modification in mRNAs, rRNAs and tRNAs. Results revealed that the ac4C modification levels were lower in the Nat10-null testes than in the WT testes. These data presumably implied a pivotal role of Nat10-catalyzed ac4C modification in spermatogonial development.
Given these findings, they next reasoned that NAT10-deposited ac4C in mRNAs might account for the deleterious effects. To this end, they deciphered the ac4C-modified mRNA landscape using an in-house optimized ac4C-RIP-seq. Results suggest that NAT10 stabilizes mRNA transcripts by depositing the ac4C mark. The ac4C-positive mRNAs that were only down-regulated upon Nat10 loss comprised a cohort of representative genes in relation to cell proliferation/differentiation and the cell cycle. Prior studies imply that ac4C modification is associated with enhanced mRNA translation; consequently, they employed an in-house optimized Ribo-seq assay and observed a greater number of ac4C-positive mRNAs with decreased RPF intensity in Nat10-deficient testes compared to WT testes. These findings underscore the critical role of NAT10 in enhancing the translation of ac4C-modified mRNA substrates, particularly those involved in cell cycle and germline development. Next, they conducted label-free quantitative mass spectrometry (MS), which identified dysregulated proteins in Nat10-deficient testes. GO enrichment analysis revealed that these proteins were closely associated with cell cycle progression, cell division, proliferation, and germline development. Collectively, these integrative analyses unambiguously showed that, at least in part, both DMRT1 and CCNA2 serve as bona fide substrates for NAT10-catalyzed ac4C modification in vivo, responsible for defective spermatogonial homeostasis. Additionally, this study identified PRRC2B as a key partner that assists NAT10 in vivo.
In this study, professor Bao’s team exploited a highly efficient, tamoxifen-inducible Nat10 KO mouse models and revealed a prominent role of NAT10 in priming a coordinated post-transcriptional repertoire in testicular spermatogonia during both first-wave spermatogenesis and adult steady-state spermatogenesis. These findings not only enhance the understanding of mechanisms underlying spermatogonial development at the epitranscriptomic level, but also provide valuable insights for future functional studies on NAT10 substrates and cofactors in other milieux.
See the article:
NAT10 primes a post-transcriptional repertoire essential for the maintenance of spermatogonial homeostasis
https://doi.org/10.1016/j.scib.2025.01.021
Journal
Science Bulletin