No abstract

Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the underlying molecular mechanism that controls alternative mRNA expression during germ cell development remains elusive. Herein, we show that hnRNPH1 is highly expressed in the reproductive system and recruits the PTBP2 and SRSF3 to modulate the alternative splicing in germ cells. Conditional knockout Hnrnph1 in spermatogenic cells causes many abnormal splicing events, thus affecting the genes related to meiosis and communication between germ cells and Sertoli cells. This is characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, which ultimately leads to male sterility. Markedly, Hnrnph1 germline-specific mutant female mice are also infertile, and Hnrnph1-deficient oocytes exhibit a similar defective synapsis and cell-cell junction as seen in Hnrnph1-deficient male germ cells. Collectively, our data support a molecular model wherein hnRNPH1 governs a network of alternative splicing events in germ cells via recruitment of PTBP2 and SRSF3.

Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did; however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RBP, FXR1 as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

Pachytene piRNA biogenesis is a hallmark of the germline, distinct from another wave of pre-pachytene piRNA biogenesis with regard to the lack of a secondary amplification process known as the Ping-pong cycle. However, the underlying molecular mechanism and the venue for the suppression of the Ping-pong cycle remain elusive. Here, we showed that a testis-specific protein, ADAD2, interacts with a TDRD family member protein RNF17 and is associated with P-bodies. Importantly, ADAD2 directs RNF17 to repress Ping-pong activity in pachytene piRNA biogenesis. The P-body localization of RNF17 requires the intrinsically disordered domain of ADAD2. Deletion of Adad2 or Rnf17 causes the mislocalization of each other and subsequent Ping-pong activity derepression, secondary piRNAs overproduced, and disruption of P-body integrity at the meiotic stage, thereby leading to spermatogenesis arrested at the round spermatid stage. Collectively, by identifying the ADAD2-dependent mechanism, our study reveals a novel function of P-bodies in suppressing Ping-pong activity in pachytene piRNA biogenesis.