PPP6R3-mediated dephosphorylation regulates mRNA translation during spermatogonial differentiation

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 (PP6), 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 EIF3C^S39 and EIF4G1^S1217 are significantly up-regulated in mutant mice. Overexpression of EIF3C^S39A and EIF4G1^S1217A mutants in Ppp6r3-cKO spermatogonial progenitor cells compensates for the deficiency of differentiation potential by upregulating translation rates of differentiation-associated mRNAs. Our findings demonstrate EIF3C and EIF4G1, as specific substrates of PPP6R3/PP6 holoenzyme, are required for translation activation during spermatogonial differentiation.