Roles And Regulatory Mechanism Of M6A Reader PRRC2A In Male Meiosis

As the damaging of male fertility by modern lifestyle and environment,the decline of male fertility has become a global issue.In China,the incidence rate of male infertility is 10%-12%.It’s urgent to protect and restore male fertility under the background of the unbalanced population structure.Spermatogenesis is a complicated developmental process,including the renewal and differentiation of spermatogonial stem cells,meiosis of spermatocytes,and dramatic morphologic changes of spermatids,which needs a precise regulatory mechanism to ensure the production of healthy sperms.N6-methyladenosine(m6A)is the most prevelant modification of eukaryotic mRNA which is catalyzed by METTL3/METTL14 and is removed by demethylase ALKBH5 and FTO.Readers recognize m6A-modified mRNA and mediate their splicing,transportation,decay,and translation,so that regulate multiple physiological and pathological processes.Previous studies showed that m6A exists in all types of male germ cells.Meanwhile,methyltransferase METTL3 and METTL14 are reported to regulate the maintenance of spermatogonial stem cells,spermatogonial differentiation,meiosis initiation,and spermiogenesis.The demethylase ALKBH5 facilitates the development of pachytene spermatocytes and spermatids;FTO regulates the cell cycle of spermatogonia cell line GC1 cells.These research showed that m6A is essential for the regulation of each stage of spermatogenesis.However,although almost twenty m6A "reader" proteins,the functional mediators of m6A,are discovered,only YTHDC1,YTHDC2,and YTHDF2 are demonstrated to regulate the development in spermatogonia and early stage of meiosis.There is no research about the regulartory mechanism of m6A during the late stage of meiosis.The current research show that m6A "reader" protein PRRC2A is highly expressed from pachytene spermatocytes to round spermatids in testes and the knockout of Prrc2a in germ cells leads to male infertility.The immunostaining and histological analysis showed that PRRC2A deficiency leads to X-Y asynapsis,impaired meiotic sex chromosome inactivation and delayed metaphase entry in prophase spermatocytes,causes chromosome misalignment and abnormal assembly of the spindle and microtubule organizing center in metaphase spermatocytes.Consequently,defective spermatocytes arrest at metaphase I and undergo massive apoptosis.Therefore,PRRC2A is essential for male meiosis.Combining RNA-seq,Ribo-seq,and PRRC2A RIP-seq,the present study reveals that PRRC2A decreases the RNA abundance or improves the translation efficiency of m6A-modified mRNAs.Specifically,PRRC2A recognizes spermatogonia-specific transcripts and downregulates their RNA abundance to promote the transcriptome transition from spermatogonia to spermatocytes,so that maintain the spermatocyte expression pattern.For proteins regulating cell division,such as WNK1 and CEP 192,PRRC2A improves the translation efficiency of their transcripts to promote the successful completion of meiosis metaphase.Further,immunoprecipitation coupled with mass spectrum experiments identify YBX1,YBX2,PABPC1,FXR1,and EIF4G3 as interacting proteins of PRRC2A.They are reported to regulate mRNA metabolism and translation in previous studies and potentially facilitate PRRC2A to regulate target mRNA.Collective,our study demonstrates the critical role of PRRC2A in male meiosis and reveals its new post-transcriptional functions,supporting the follow-up research on the mechanism of PRRC2A and other m6A reading proteins and providing the theoretical foundation for the treatment of related diseases including male infertility.