Rna-Binding Protein Maca Is Crucial For Gigantic Male Fertility Factor Gene Expression, Spermatogenesis, And Male Fertility, In Drosophila

Author summary Sperm is produced from germline cells via the process called spermatogenesis, during which a special gene expression program dedicated to this process operates. In fruit fly Drosophila, extremely large male fertility factor genes on Y-chromosome containing mega-base-sized introns are expressed only during spermatogenesis, and their successful expression is crucial for spermatogenesis. However, expressing such large genes must be very challenging for cells and the molecular mechanisms that ensure their successful expression remain unknown. In this study, we identified a novel RNA-binding protein encoded in the gene CG5213 that is required for this process. We created mutant flies lacking this protein using a genome-editing technique, and investigated them using genetic, microscopic, molecular, transcriptomic, and proteomic approaches. We found that this RNA-binding protein is crucial for successful expression of the extremely large male fertility factor genes, spermatogenesis, and male fertility. We named the CG5213 gene maca, after the plant maca grown in the high Andes mountains whose root has been traditionally believed and used to increase sperm quality and promote male fertility. By identifying the novel RNA-binding protein Maca, our studies give significant insight into how the special gene expression program operates during spermatogenesis.During spermatogenesis, the process in which sperm for fertilization are produced from germline cells, gene expression is spatiotemporally highly regulated. In Drosophila, successful expression of extremely large male fertility factor genes on Y-chromosome spanning some megabases due to their gigantic intron sizes is crucial for spermatogenesis. Expression of such extremely large genes must be challenging, but the molecular mechanism that allows it remains unknown. Here we report that a novel RNA-binding protein Maca, which contains two RNA-recognition motifs, is crucial for this process. maca null mutant male flies exhibited a failure in the spermatid individualization process during spermatogenesis, lacked mature sperm, and were completely sterile, while maca mutant female flies were fully fertile. Proteomics and transcriptome analyses revealed that both protein and mRNA abundance of the gigantic male fertility factor genes kl-2, kl-3, and kl-5 (kl genes) are significantly decreased, where the decreases of kl-2 are particularly dramatic, in maca mutant testes. Splicing of the kl-3 transcripts was also dysregulated in maca mutant testes. All these physiological and molecular phenotypes were rescued by a maca transgene in the maca mutant background. Furthermore, we found that in the control genetic background, Maca is exclusively expressed in spermatocytes in testes and enriched at Y-loop A/C in the nucleus, where the kl-5 primary transcripts are localized. Our data suggest that Maca increases transcription processivity, promotes successful splicing of gigantic introns, and/or protects transcripts from premature degradation, of the kl genes. Our study identified a novel RNA-binding protein Maca that is crucial for successful expression of the gigantic male fertility factor genes, spermatogenesis, and male fertility.