NSUN7 is an RNA:m5C catalytically inactive protein that regulates sperm flagellum assembly

Defects in Nsun7, a member of the main RNA:m5C modifying enzymes, known as the NOL1/NOP2/Sun domain (NSUN) family, cause male infertility. The NSUN family includes NSUN1-7, all possessing conserved motifs IV and VI essential for RNA:m5C formation. However, due to the controversy regarding the catalytic activity and RNA substrates of NSUN7, the pathogenic mechanism underlyingof NSUN7-related male infertility remains unclear. Here, we showed that NSUN7 is predominantly enriched in adult mouse testes, particularly in elongated spermatids. Through m5C mass spectrometry detection and bisulfite sequencing, we found that knockout of Nsun7 has no impact on testicular RNA:m5C modification. Mechanistically, NSUN7 loses its ability to bind S-adenosylmethionine (SAM) due to sequence variations in motif IV compared towith the conserved residues observed in NSUN1-6. Notably, the key SAM-binding Asp in motif IV is replaced by Leu in NSUN7, thereby abolishing SAM binding. Nsun7 deficiency impairs sperm progressive motility, accompanied by defects in axonemes and mispositioning of longitudinal columns. Single-cell RNA sequencing showed that knockout of Nsun7 results in a significantly decrease in the levels of a cohort of mRNAs related to cilium organization in elongated spermatids. Collectively, our study demonstrateds that NSUN7 is the only RNA:m5C catalytically inactive member in the NSUN family, and that it regulates sperm flagellum assembly by modulating the expression of cilium organization-associated genes. Overall design: ultrafast bisufite-seq profiling of m5C modification levels in mRNA, large RNA, and small RNA from WT and Nsun7 KO adult mouse testes