Positioning of sperm tail longitudinal columns depend on Nsun7, an RNA binding protein destabilizing elongated spermatid transcripts (CLIP-Seq)

Spermatozoid’s flagella assemble in transcriptionally silent spermatids and thus depends on post-transcriptional regulation of gene expression. Mutations in Nsun7 gene are known to cause male infertility in human and mice. We identified m5C-specific Nsun7 RNA methyltransferase as a protein present in elongated spermatids and interacting with RNAs specific for this type of spermatozoid’s precursor cells. Inactivation of Nsun7 gene in mice leads to upregulation of its RNA interactors, thus indicating that Nsun7 downregulates a set of RNAs in the elongated spermatids. A physiologic consequence of Nsun7 gene knockout is male infertility, which is mechanistically explained by the observed mispositioning of longitudinal columns relative to the axonemal microtubular doublets leading to motility defect. In order to identify the binding site of m5C RNA methyltransferase Nsun7, we performed UV crosslinking of Nsun7 with its RNA targets, followed by further co-immunoprecipitation of the obtained covalent complexes. To obtain a sufficient material, tissue from 30 wild-type and 30 HA-tagged Nsun7 mice was used.