LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice.

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.

piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis.

The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice, with MIWI initially assembling in the IMC for piRNA processing before translocating to the CB for functional deployment. The regulatory mechanism underpinning MIWI translocation, however, has remained elusive. We unveil that piRNA loading is the trigger for MIWI translocation from the IMC to CB. Mechanistically, piRNA loading facilitates MIWI release from the IMC by weakening its ties with the mitochondria-anchored TDRKH. This, in turn, enables arginine methylation of MIWI, augmenting its binding affinity for TDRD6 and ensuring its integration within the CB. Notably, loss of piRNA-loading ability causes MIWI entrapment in the IMC and its destabilization in male germ cells, leading to defective spermatogenesis and male infertility in mice. Collectively, our findings establish the critical role of piRNA loading in MIWI translocation during spermatogenesis, offering new insights into piRNA biology in mammals.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility.

PIWI-clade proteins harness piRNAs of 24-33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR super-complex for translational activation. Importantly, we identify a c.1108C>T (p.R370W) mutation of HIWI (human PIWIL1) in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility.