NSUN7 is a catalytically inactive RNA m^5C methyltransferase essential for sperm flagellum assembly

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: minor_revision

Abstract
RNA 5-methylcytosine (m5C) is a widespread RNA modification mainly catalyzed by the NSUN family, whose dysfunction is linked to various diseases. Among NSUN1-7, all possessing motifs IV and VI essential for RNA m5C formation, NSUN7’s catalytic activity and physiological role remain unclear, despite its known association with male infertility. Here, we show that NSUN7 is predominantly enriched in adult mouse testes, particularly in elongated spermatids. Through transcriptome-wide m5C mass spectrometry and ultrafast bisulfite sequencing, we found that Nsun7 knockout has no impact on RNA m5C modification. Mechanistically, compared to NSUN1-6, NSUN7 lacks S-adenosylmethionine (SAM) binding ability due to sequence variations in motif IV, where the key SAM-binding Asp in motif IV is replaced by Leu, thereby abolishing its catalytic activity. Nsun7 deficiency impairs sperm progressive motility, accompanied by defects in axonemes and mispositioning of longitudinal columns. Single-cell RNA sequencing shows that Nsun7 knockout decreases the levels of a cohort of mRNAs related to cilium organization in elongated spermatids. Collectively, our findings resolve the long-standing question of NSUN7’s enzymatic role and reveal a non-catalytic regulatory function, providing a more complete understanding of NSUN family biology.

Introduction
Currently, more than 170 types of chemical modifications have been found on various RNA species1, greatly expanding the functional diversity of RNA. RNA modifications not only affect the structure of RNA2, but also regulate RNA localization3,4, processing5,6, stability7,8, and participate in multiple biological processes, including protein translation9, nutrient metabolism10, and stress responses11. Notably, dysregulation of RNA modifications is closely related to a range of human diseases12,13, such as neurological disorders14, metabolic syndromes15, and male infertility16.

Among these diverse RNA modifications, 5-methylcytosine (m5C) is one of the most prevalent RNA modifications, and is of particular interest due to its abundance across a wide variety of RNA species, including tRNA, rRNA, and mRNA1,17,18. RNA m5C is catalyzed by DNMT2, a member of the DNA methyltransferase (DNMT) family, and by the NOL1/NOP2/Sun domain (NSUN) methyltransferase family17. Both DNMT2 and the NSUN family use S-adenosylmethionine (SAM) as the methyl donor to catalyze RNA m5C modification, highlighting the critical role of SAM binding in the catalytic process. In higher eukaryotes, the NSUN family comprises seven members, named NSUN1-7. All seven members have the canonical NSUN domain, which contains both an RNA m5U-like motif (motif VI) and a DNA m5C-like motif (motif IV)19,20. The Cys residue in motif VI serves as a nucleophile, and the Cys residue in motif IV acts as a general base to initiate product release19,20. The RNA m5C catalytic activity of the NSUN domain also relies on the collaboration of several amino acid residues located within these two motifs, such as Asp, Ala, and Pro in motif IV19,20. Structural and functional analyses revealed that Asp in motif IV is indispensable for RNA m5C formation, as substitution of Asp with Asn in motif IV abolishes both SAM binding and enzymatic activity of NSUN6, ultimately leading to autosomal recessive intellectual disability21.

To date, the RNA substrates of most NSUN family members have been extensively characterized. NSUN1 is responsible for a specific m5C modification site on cytoplasmic 28S rRNA22,23. NSUN2 can modify mRNA, vault RNA, and cytoplasmic and mitochondrial tRNA24–27. NSUN3 catalyzes methylation of mitochondrial tRNA28,29. NSUN4 was described as methylating mitochondrial 12S rRNA and other mitochondrial RNAs30,31. NSUN5 modifies cytoplasmic 28S rRNA and mRNA32,33. NSUN6 is able to methylate cytoplasmic tRNA and mRNA20,34–36. However, the enzymatic activity and RNA substrates of NSUN7 remain controversial37,38.

The defects in Nsun7 result in impaired sperm motility and male infertility in mice39. In humans, mutations such as the C26232T transition, T26248G transversion in exon 7, and an A11337 deletion are tightly associated with reduced sperm motility, and can serve as infertility markers in Iranian asthenospermic men40,41, highlighting the importance of NSUN7 in spermatogenesis and sperm maturation. Moreover, NSUN7 is closely linked to the development of various cancers and neurological diseases38,42–44. Nevertheless, the unresolved controversy surrounding the RNA m5C catalytic activity and RNA substrates of NSUN7 hinders progress in elucidating its physiological and pathological functions.

During spermiogenesis45,46, spermatogonia undergo mitosis to produce primary spermatocytes, which then enter meiosis I to form secondary spermatocytes. Following meiosis II, these cells differentiate into round spermatids. Through a series of remodeling and maturation steps, round spermatids transform into elongated spermatids. Subsequently, elongated spermatids enter the epididymis, and further mature into fully functional spermatozoa. Central to the spermatozoatail is a microtubule-based axoneme structure composed of a characteristic ‘9+2’ arrangement of microtubules47,48. Structurally, spermatozoa consist of several distinct parts: the head, which contains the nucleus and is capped by the acrosome; the neck, which connects the head to the tail; and the tail, also known as the flagellum, subdivided into three main segments: the midpiece, principal piece, and endpiece49. The midpiece houses numerous mitochondria arranged in a spiral formation, forming the mitochondrial sheath that provides energy for sperm motility. The principal piece constitutes the majority of the tail length and contains two longitudinal columns (LCs) within the fibrous sheath. Finally, the endpiece, the shortest segment of the tail, continues the extension of the axoneme. Together, these components ensure the ability of spermatozoa to navigate through the female reproductive tract and achieve fertilization. Defects in sperm structure or mitochondrial dysfunction commonly lead to sperm motility disorders and male infertility50,51.

In this study, we showed that NSUN7 is primarily localized in adult mouse testes, especially in elongated spermatids. Through m5C mass spectrometry (MS) and ultrafast bisulfite sequencing analyses of different types of RNA from testicular tissues, we unexpectedly found that Nsun7 knockout has no impact on the RNA m5C modification level. Mechanistically, we showed that NSUN7 is incapable of binding SAM, indicating that NSUN7 is an RNA m5C catalytically inactive protein. This catalytic deficiency arises from sequence variations in motif IV of NSUN7 compared to the conserved residues observed in NSUN1-6, particularly the substitution of the critical SAM binding residue Asp with Leu in this region, thereby abolishing SAM binding. In Nsun7 knockout mice, the progressive motility of sperm was severely impaired, accompanied by axonemal defects and mispositioning of LCs. Moreover, single-cell RNA sequencing of testes revealed that Nsun7 knockout resulted in a significant downregulation of a cohort of mRNAs in elongated spermatids, particularly transcripts associated with cilium organization. In summary, our findings demonstrate that NSUN7 is the only RNA m5C catalytically inactive member in the NSUN family, and reveal its critical role in regulating sperm axoneme structure assembly and maintaining the position of LCs.

Results
NSUN7 is an evolutionarily conserved protein in vertebrates and exhibits testis-specific enrichment in the mouse

Using sequence alignment analysis (Supplementary Fig. 1), we found that NSUN7 is evolutionarily conserved among vertebrates, and exhibits high sequence similarity across species, including human, mouse, Bos taurus, Macaca fascicularis, Gallus gallus, Xenopus laevis, and Maylandia zebra. Importantly, NSUN7 proteins in different species retain the two conserved NSUN family motifs: an RNA m5U-like motif (motif VI) and a DNA m5C-like motif (motif IV).

To determine the expression profile of Nsun7, we conducted RT-qPCR using multiple tissues from adult 12-week-old mice. Our results showed that the mRNA expression of Nsun7 is predominantly enriched in mouse testes (Fig. 1A). Consistently, NSUN7 protein was clearly detected in adult mouse testes using a homemade NSUN7-specific antibody (Fig. 1B). In addition, we isolated various reproductive tissues of female mice, including the ovary, fallopian tube, uterus, and vagina. In contrast, NSUN7 protein was too low to be detected in these tissues with testis serving as the positive control (Supplementary Fig. 2). Further analysis of Nsun7 mRNA expression at different developmental stages of mouse testes showed that Nsun7 transcription begins at approximately postnatal day 28 (Fig. 1C), and this expression pattern was also confirmed at the protein level (Fig. 1D). Notably, postnatal day 28 corresponds well to the developmental stage at which elongated spermatids initially appear in mouse testes52, suggesting that NSUN7 may play a functional role during the late stages of spermatid differentiation. Since Nsun7 is highly expressed in adult mouse testes, all subsequent experiments used 12-week-old mice unless otherwise specified.

To investigate the subcellular localization of NSUN7, we expressed the gene encoding C-terminally Flag-tagged NSUN7 (NSUN7-Flag) in HEK293T cells and performed immunolabeling. Immunofluorescence analysis revealed that NSUN7 is primarily localized in the cytoplasm, with minor nuclear presence (Supplementary Fig. 3A). To understand the physiological function of NSUN7 and explore its role in sperm development, we generated a Nsun7 knockout mouse model using the CRISPR-Cas9 system (Fig. 1E), and subsequently evaluated NSUN7 protein levels in wild-type (WT), heterozygous mutant (Nsun7+/−), and homozygous mutant (Nsun7−/−) mouse testes (Fig. 1F). To examine the subcellular localization of NSUN7 in adult mouse testes, we performed immunofluorescence staining and observed that NSUN7 protein was localized in the testicular lumen (Fig. 1G). Further analysis showed that NSUN7 colocalized with the microtubule protein acetylated tubulin (Ac-Tub) (Supplementary Fig. 3B), suggesting that NSUN7 specifically localizes to the flagella of elongated spermatids.

Thus, our results showed that NSUN7 is predominantly enriched in adult mouse testis, with specific localization to the flagella of elongated spermatids, suggesting a potential role for NSUN7 in the late stages of sperm development.

Knockout of Nsun7 does not affect m5C levels on different types of RNA in the mouse testes assayed by RNA MS

As a member of the NSUN family, NSUN7 harbors the canonical NSUN domain. Considering that the NSUN domain of NSUN7 retain the two catalytic Cys residues essential for RNA m5C formation (Supplementary Fig. 1), we intended to investigate whether NSUN7 possesses RNA m5C methyltransferase activity and to decipher its impact on RNA m5C modification. We isolated different types of RNA from adult WT and Nsun7−/− mouse testes, and detected m5C modification levels through UPLC-MS/MS analysis. Total RNA samples were fractionated into two different pools: RNA smaller than 200 nt and RNA larger than 200 nt (Fig. 2A). We subsequently isolated 18S and 28S rRNA from RNA larger than 200 nt (Fig. 2A). For each RNA pool, we quantified the m5C levels by UPLC-MS/MS and normalized their abundance to the abundance of unmodified nucleoside A. Unexpectedly, no significant differences were observed in m5C modification levels in total RNA, RNA smaller than 200 nt, and RNA larger than 200 nt between WT and Nsun7−/− mouse testes. Moreover, we also found that knockout of Nsun7 had no significant effect on the m5C modification level of 28S rRNA from testes (Fig. 2B). For 18S rRNA, we did detect m5C modifications in either WT or Nsun7−/− mouse testes (Fig. 2B). On the other hand, we enriched mRNA from testicular total RNA using oligo dT beads to examine m5C modification levels (Fig. 2C). However, the m5C levels in enriched mRNA samples were below the detection limit of our MS analysis (Fig. 2D), likely due to the inherently low abundance of m5C modifications on mRNA.

Given that NSUN7 is mainly localized in the elongated spermatids of testes (Fig. 1G), and that Nsun7 mRNA is probably highest in round and elongated spermatids53, we isolated haploid spermatids including round and elongated spermatids from adult WT and Nsun7−/− mouse testes using a method based on the principle of STA-PUT54,55, and extracted intracellular RNAs to assess the effect of NSUN7 on RNA m5C levels. Total RNA samples from haploid spermatids were fractionated into two pools: RNA smaller than 200 nt and RNA larger than 200 nt (Fig. 2E). m5C modification levels were detected in these RNA fractions from WT and Nsun7−/− haploid spermatids. Consistent with the results from whole testes, knockout of Nsun7 had no obvious influence on RNA m5C modifications in either RNA fraction from haploid spermatids (Fig. 2F).

Together, these RNA MS results show that knockout of Nsun7 does not affect m5C modification levels in different types of RNA from mouse testes or testicular haploid spermatids.

UBS-sequencing reveals that knockout of Nsun7 has no impact on testicular mRNA m5C modification nor on tRNA or rRNA

Given the inherent limitations of UPLC-MS/MS in detecting low-abundance m5C modification on mRNA56, to verify the impact of NSUN7 on mRNA m5C modification, we isolated mRNA from adult WT and Nsun7−/− mouse testes and performed ultrafast bisulfite sequencing (UBS-seq)57 to profile m5C modification sites and levels on mRNA (Fig. 3A). In WT testes, we identified 251 high-confidence mRNA m5C sites (Fig. 3B; Supplementary Data 2), with a median methylation level of 0.1 (Fig. 3B), consistent with the low abundance of mRNA m5C modification previously indicated by our UPLC-MS/MS results. Notably, comparative analysis revealed that knockout of Nsun7 neither led to the emergence of additional modification sites, nor significantly altered mRNA m5C modification levels, suggesting that knockout of Nsun7 had no effect on m5C modification sites or their modification levels (Fig. 3C). Furthermore, we extracted the top ten mRNAs with the highest sequencing coverage, including Asrgl1, Insl3, Tcp10c, Ubb-ps, Pabpc1, Ldhal6b, Spata18, Ybx3, Atp8b3, Hk1. In all cases, the sequencing results showed no obvious changes in RNA m5C levels on these mRNAs (Fig. 3D).

To comprehensively validate the impact of NSUN7 on RNA m5C modifications across the transcriptome, we separately isolated RNA larger than 200 nt and RNA smaller than 200 nt from WT and Nsun7−/− mouse testes, and conducted UBS-seq analyses. In large RNAs, the highest sequencing coverage was observed for 28S rRNA, which exhibited m5C modifications at positions 3438 and 4099 (Fig. 3E, F and Supplementary Data 3). Notably, Nsun7 deficiency did not affect the m5C levels at either site (Fig. 3E, F).

In small RNAs, we identified 192 high-confidence m5C sites in WT testes, with a median modification level of 0.8, consistent with the high m5C levels in small RNAs detected by UPLC-MS/MS. Remarkably, Nsun7 knockout neither generated additional modification sites nor significantly affected the m5C levels of these 192 sites (Fig. 3G, H and Supplementary Data 4). The small RNA with the highest sequencing coverage was tRNA-Glu-TTC-1-1, exhibiting nearly 100% m5C modification at positions 49 and 50, which showed no difference in Nsun7−/− mouse testes compared to WT controls (Fig. 3I).

For mitochondrial RNA, we identified a total of nine m5C modification sites, located on 12S rRNA and six tRNAs. Knockout of Nsun7 did not affect the m5C modification sites or their modification levels (Fig. 3J and Supplementary Data 5). We detected m5C modification at position 842 on 12S rRNA (position 911 at mitochondrial genome), which remained unchanged in Nsun7−/− mouse testes compared to WT controls (Fig. 3K). Additionally, we detected eight m5C modification sites across six mitochondrial tRNAs (mt-tRNA-Ser2, mt-tRNA-His, mt-tRNA-Leu1, mt-tRNA-Leu2, mt-tRNA-Met, mt-tRNA-Pro), exhibiting distinct modification levels. We found that knockout of Nsun7 had no impact on m5C modification levels on these mitochondrial tRNAs (Fig. 3L).

In summary, combining UPLC-MS/MS and UBS-seq on comprehensive transcriptome-wide profiling of m5C modifications, our results demonstrate that NSUN7 is dispensable for m5C modifications in mouse testes across various RNA species, as neither the number of methylation sites nor their modification levels are affected by Nsun7 knockout.

NSUN7 is catalytically inactive for RNA m5C methylation due to a lack of SAM-binding capability

The seven NSUN members, NSUN1-7, all contain the canonical NSUN domain, consisting of two conserved motifs IV and VI. Our previous work on the structure of NSUN6/tRNA/SAM complex (PDB:5WWS) demonstrated that the amino acid residues “DAPCS” in motif IV are critical for RNA m5C formation20. Interestingly, “DAPCS” in motif IV is highly conserved in NSUN1-6, while it is replaced with “LPRCS” in NSUN7 (Fig. 4A), suggesting that NSUN7 may have a different catalytic mechanism or enzymatic activity.

Sequence analysis and structure prediction demonstrated that the 1–188 amino acid residues form the N-terminal domain of mouse NSUN720, while 208–518 amino acid residues constitute the NSUN domain (Fig. 4B, C). The C-terminal extension domain is predicted to be intrinsically disordered (Fig. 4B) by IUPred3 software58. Our previous study showed that the side chain carboxyl group of Asp in motif IV forms hydrogen bonds with the N3 and N4 nitrogen atoms of the targeted cytosine (C), as well as with the amino group of SAM20. These interactions precisely orient the base in the active site and facilitate bond formation by transient protonation of the endocyclic N3 of C (Fig. 4B). When Asp is mutated to Ala, NSUN6 fails to bind SAM and completely loses its methyltransferase activity20. Furthermore, a missense mutation of Asp to Asn in motif IV abolishes SAM-binding capacity, resulting in a loss of enzymatic activity of NSUN6 and contributing to autosomal recessive intellectual disability21, indicating the indispensable role of Asp in motif IV for RNA m5C catalysis. In mouse NSUN7, the corresponding substitution of this Asp with Leu in motif IV may disrupt these precise interactions (Fig. 4D), potentially altering the enzymatic activity of NSUN7 compared to other members of the family.

To verify this hypothesis, we attempted to purify the full-length mouse NSUN7 protein to test its ability to bind SAM. However, despite optimizing various conditions, we found it difficult to obtain mouse NSUN7 protein in its full-length form. We therefore purified the NSUN domain of mouse NSUN7, which was obtained with high purity and confirmed to exist as a monomer in solution (Fig. 4E, F). Subsequently, we performed ITC to evaluate its SAM-binding capability. Intriguingly, we found that the NSUN domain of mouse NSUN7 does not bind SAM at all (Fig. 4G).

To investigate whether the substitution of Asp with Leu in motif IV is the major cause of NSUN7 lacking SAM-binding capacity, we reverted Leu378 to Asp in the NSUN domain of mouse NSUN7 and assessed the SAM-binding capacity of this mutant protein. Both wild-type and L378D mutant NSUN domains of mouse NSUN7 were purified with high purity (Supplementary Fig. 4A), and their SAM-binding capacities were examined using ITC (Supplementary Fig. 4B–D). During the initial injections of SAM into the L378D mutant NSUN domain, distinct exothermic signals were observed. Subsequently, at the 5th and 8th injections, distinct exothermic and endothermic peaks were detected, which probably resulted from SAM-induced conformational changes in the protein during titration. In the subsequent SAM titrations, the exothermic signals gradually weakened and eventually stabilized. In contrast, the wild-type NSUN domain showed no detectable heat changes throughout the titration. These results suggest that the L378D mutant NSUN domain is capable of binding SAM.

A similar substitution of Asp with Leu in motif IV was also observed in human NSUN7 (Supplementary Fig. 5A–D). Sequence alignment and AlphaFold structure prediction showed that the NSUN domain of human NSUN7 comprises 200–513 amino acid residues (Supplementary Fig. 5B, C)59. We then purified the NSUN domain of human NSUN7 (Supplementary Fig. 5E, F) and tested its SAM-binding affinity. Consistent with the result of mouse NSUN7, the NSUN domain of human NSUN7 also failed to bind SAM (Supplementary Fig. 5G).

Collectively, our results indicate that although NSUN7 maintains the canonical NSUN domain, sequence variations in motif IV and the critical SAM-binding residue change from Asp to Leu render it incapable of SAM binding and consequently inactive for RNA m5C methylation, suggesting that NSUN7 is an RNA m5C catalytically inactive member of the NSUN family.

Nsun7 deficiency causes a sperm progressive motility defect and male infertility

Considering the correlation between NSUN7 and male infertility, we generated adult WT, Nsun7+/−, and Nsun7−/− mice to explore the role of NSUN7 in male reproduction. Adult Nsun7−/− mice appeared normal and healthy, and were indistinguishable from their WT and heterozygous littermates (Fig. 5A). The body and testis weights of Nsun7−/− mice were similar to those of littermate controls (Fig. 5B–D). To test fertility, we housed individual males (WT and Nsun7−/−) with WT females, and individual females (Nsun7−/−) with WT males. Strikingly, we found that all tested Nsun7−/− male mice were completely sterile, while Nsun7−/− female mice exhibited normal fertility (Fig. 5E), highlighting a specific requirement for NSUN7 in male reproduction. To examine whether Nsun7 deficiency caused any defect in spermatogenesis, we performed histological analysis of testes and epididymides. Similar to WT, all germ cell populations (spermatogonia, spermatocytes, round spermatids, and elongated spermatids) appeared normal in Nsun7−/− testes (Fig. 5F). In addition, numerous spermatozoa could be observed in Nsun7−/− epididymides (Fig. 5G). Thus, these findings indicated that NSUN7 has no impact on the process of spermatogenesis.

To reveal the cause of infertility in Nsun7−/− male mice, we initially examined spermatozoa collected from the cauda epididymides. However, no obvious abnormalities were observed in sperm morphology, acrosome structure, or mitochondrial sheath organization in Nsun7−/− sperm compared to WT (Fig. 5H). Subsequently, we examined sperm count and motility using computer-assisted sperm analysis (CASA). Although sperm counts and the proportion of motile sperm in Nsun7−/− mice were similar to those in controls (Fig. 5I, J), the percentage of progressively motile sperm dramatically decreased in Nsun7−/− samples (Fig. 5K), suggesting that spermatozoa from Nsun7−/− cauda epididymides showed severely impaired progressive motility. To analyze the fertilizing capacity of Nsun7−/− spermatozoa, we conducted in vitro fertilization (IVF) assays using WT oocytes. Quantitative analysis revealed severe impairment of fertilization competence in Nsun7−/− sperm, with only 22 out of 271 oocytes successfully fertilized (8.1% fertilization rate; Fig. 5L and Supplementary Fig. 6). These results indicated that Nsun7−/− male mice are infertile due to severely impaired sperm progressive motility.

Together, our results demonstrate that Nsun7 is a critical gene for the development of sperm progressive motility and male fertility.

Nsun7 deficiency disrupts axonemal integrity and causes mispositioning of LCs in spermatozoa

To assess the effect of NSUN7 in sperm, we conducted ultrastructural detection of epididymal mature spermatozoa from WT and Nsun7−/− mice using transmission electron microscopy (TEM) (Fig. 6A). We found that both WT and Nsun7−/− sperm heads exhibited condensed structures and showed no obvious differences (Fig. 6B). These observations suggested that Nsun7 knockout does not affect the structure of the sperm head.

Considering that the maintenance of mitochondrial structure and function is critical for spermatozoa motility, we next investigated whether Nsun7 deficiency could affect mitochondrial function. Mitochondrial membrane potential (MMP), a sensitive biomarker reflecting mitochondrial status, was measured using JC-1 staining and revealed no significant differences between Nsun7−/− and WT spermatozoa (Supplementary Fig. 7A). Since mitochondria are the predominant source of reactive oxygen species (ROS), we also measured ROS levels using 7-di-chlorodihydrofluorescein diacetate (DCFH-DA) staining. Notably, ROS production in Nsun7−/− spermatozoa had no significant changes compared to WT (Supplementary Fig. 7B). Together, these results showed that knockout of Nsun7 does not impair mitochondrial function in mature sperm.

We further inspected the flagella cross-sections of WT and Nsun7−/− spermatozoa. In the midpiece, well-defined outer dense fibers (ODFs) and the axoneme consisting of the typical ‘9+2’ microtubules were both observed in WT and Nsun7−/− spermatozoa, whereas the Nsun7−/− spermatozoa displayed more axonemal defects, including missing microtubules (Fig. 6C). Notably, the incidence of axonemal defects increased progressively from the midpiece to the principal piece and endpiece regions (Fig. 6D).

Additionally, we also observed mispositioning of LCs within the fibrous sheath of Nsun7−/− spermatozoa (Fig. 6E). In WT sperm, LCs are typically attached to outer microtubule doublets 3 and 8. However, in Nsun7−/− sperm, only 25% of LCs showed normal attachment, while approximately 72% of LCs were connected to microtubule doublets 2 and 8 (Fig. 6F). Microtubules provide necessary mechanical support through their organized arrangement, serving as the foundation for flagellar movement. LCs further stabilize this structure, helping maintain the proper alignment and function of the microtubules. As both axonemal microtubules and LCs are essential for flagellar stability and motility60, these observed defects likely contribute to the severely impaired progressive motility of Nsun7−/− spermatozoa.

Thus, our results demonstrate that knockout of Nsun7 does not affect mitochondrial function, but leads to severe flagellar microtubule defects and mispositioned longitudinal columns, which are critical for normal sperm motility.

Nsun7 depletion downregulates a cohort of mRNAs associated with cilium organization in elongated spermatids

To investigate the molecular mechanisms of Nsun7 in sperm axonemal assembly, we performed single-cell RNA sequencing (scRNA-seq) of adult testes from WT and Nsun7−/− mice, given the testis-specific enrichment of NSUN7. A complex testicular cell type was unsupervisedly visualized using the uniform manifold approximation and projection (UMAP) algorithm (Fig. 7A), and ten cell types were identified according to the expression of canonical cell-type-specific markers as reported previously (Fig. 7B)61. These included six somatic cells, Leydig, Sertoli cells, macrophage cells, smooth muscle cells, other immune cells, and endothelium cells, and four germ cells, including spermatogonia, spermatocytes, round spermatids, and elongated spermatids.

We then investigated whether knockout of Nsun7 induced changes in cellular or molecular events in different cell populations. scRNA-seq revealed that Nsun7 mRNA is expressed in spermatocytes, round spermatids, and elongated spermatids (Fig. 7C), consistent with published datasets53. We identified differentially expressed genes (DEGs) in these three Nsun7-expressing germ cell types (14 DEGs in spermatocytes, 12 DEGs in round spermatids, and 129 DEGs in elongated spermatids, Supplementary Data 6). Notably, elongated spermatids exhibited the largest number of DEGs.

In Nsun7−/− elongated spermatids, 128 genes were significantly downregulated, and 1 gene was significantly upregulated compared to WT (Fig. 7D and Supplementary Data 6). Gene Ontology (GO) enrichment analysis of these 128 DEGs showed that knockout of Nsun7 leads to downregulation of a series of genes that were most enriched in “cilium organization” biological process and “motile cilium” cellular component (Fig. 7E), aligning with the observed structural defects in sperm flagella. Intriguingly, among the enriched cilium organization-associated genes, the expression levels of Tuba3a and Tuba3b showed the most significant changes (Fig. 7D and Supplementary Data 6). To verify the above results, we isolated elongated spermatids from WT and Nsun7−/− testes, and detected the expression levels of DEGs through RT-qPCR analysis. Our results showed that the mRNA levels of several well-studied genes involved in cilium organization, including Cfap2062, Cfap5263, Cfap4564, Wdr3565, Tuba3a66,67, and Tuba3b66,67 were certainly downregulated in Nsun7−/− elongated spermatids, while control genes Psmf1, Sord, Capza3, and Mlf1 showed no changes (Fig. 7F).

To further validate the role of NSUN7 in sperm flagella assembly, we performed data-independent acquisition (DIA) proteomic MS analysis to examine global translational changes in haploid spermatids from Nsun7−/− testes (Supplementary Data 7). Compared to WT controls, 26 significantly downregulated and 19 upregulated proteins were identified in Nsun7−/− haploid spermatids (Supplementary Fig. 8A). GO analysis of genes corresponding to the downregulated proteins showed significant enrichment in cytoskeleton-related pathways (Supplementary Fig. 8B). The cytoskeleton, comprising actin filaments, microtubules, intermediated filaments, and septins, plays a fundamental role in sperm flagella organization and cellular functions including cell motility and intracellular trafficking68. The downregulation of cytoskeletal proteins in Nsun7−/− haploid spermatids further supports a pivotal role for NSUN7 in sperm flagella assembly.

To further explore the molecular characteristics of NSUN7 and potential mechanisms underlying its role, we examined the effect of RNA on NSUN7. Treatment of mouse testis lysates with RNase A markedly decreased the endogenous NSUN7 protein level (Supplementary Fig. 9A). Consistently, ectopic expression of human NSUN7 in cultured cells showed that RNase A treatment reduced NSUN7 protein abundance (Supplementary Fig. 9B). Our results suggest that the association of NSUN7 with RNA contributes to maintaining its protein stability.

We next investigated how disease-associated mutations in NSUN7 affect its properties and function. Therefore, we generated plasmids carrying the reported human male infertility-associated point mutation p.S308A (corresponding to C26232T and T26248G in exon 7), and a deletion (A11337 deletion in exon 4), which introduces a frameshift resulting in a truncated protein of 156 amino acids40,41, and expressed them in cultured cells. The p.S308A mutant exhibited markedly reduced NSUN7 protein level compared with the wild-type protein, whereas no detectable expression was observed for the A11337-deletion mutant (Supplementary Fig. 9C). Our results suggest that these infertility-associated mutations reduce NSUN7 protein levels and may affect its function. Collectively, our study reveals that NSUN7 is specifically enriched in adult mouse testes, particularly in elongated spermatids. NSUN7 is the only RNA m5C catalytically inactive member of the NSUN family due to sequence variations in motif IV, especially the key SAM-binding Asp in motif IV is replaced by Leu in NSUN7, thereby abolishing SAM binding. Nsun7 deficiency impairs sperm progressive motility, thus leading to male infertility. While mitochondrial function is not affected, Nsun7 knockout causes defects in axoneme structure and mispositioning of longitudinal columns. Thus, NSUN7 plays a critical role in regulating sperm axoneme structure and maintaining the position of LCs by modulating the expression of cilium organization-associated genes in elongated spermatids (Fig. 8).

Methods
All animals were housed and treated according to the procedures approved by the Animal Ethics Committee of ShanghaiTech University, and all animal experiments were done in compliance with ethical guidelines and the approved protocols (ethics no: 20231030001).

Materials

Cytidine (C), adenosine (A), guanosine (G), uridine (U), m5C, 5’-guanosine monophosphate (GMP), ammonium acetate (NH4OAc), Tris-base, β-mercaptoethanol (β-Me), benzonase, pyrophosphate, and phosphodiesterase I were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Tris-HCl, tryptone, yeast extract, MgCl2, NaCl, ATP, CTP, GTP, UTP, and isopropyl-D-thiogalactoside (IPTG) were purchased from Sangon Biotech (Shanghai, China). DNA fragment rapid purification, plasmid extraction kits, HiScript III RT SuperMix for qPCR, and ChamQ Universal SYBR QPCR Master Mix were purchased from Vazyme Biotech (Nanjing, China). KOD-plus mutagenesis kit and KOD-plus-neo Kit were from TOYOBO. Dynabeads protein G, lipofectamine 2000, and RNAiMAX transfection reagent, TRIzol, Bacterial alkaline phosphatase, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution pH 7.0, T4 DNA ligase, 4’,6-diamidino-2-phenylindole (DAPI), ribonuclease inhibitor, all restriction endonucleases, polyvinylidene fluoride (PVDF) membranes and chemiluminescent substrates were obtained from Thermo Scientific (Waltham, MA, USA). EZ-trans was purchased from Life-ilab (Shanghai, China). Tissue-Tek O.C.T., bovine serum albumin (BSA), Collagenase I, and DNase I were purchased from Yeasen (Shanghai, China). TYH (Toyoda, Yokoyama, Hoshi) capacitation medium was purchased from Aibei (Nanjing, China). Enhanced immunostaining permeabilization buffer, immunostaining blocking buffer, and hematoxylin and eosin staining kit were purchased from Beyotime (Shanghai, China). Qproteome nuclear protein kits and Ni2+-NTA Superflow resin were purchased from Qiagen Inc. (Hilden, Germany). SAM was purchased from New England BioLabs, Inc. PCR primers were synthesized by BioSune (Shanghai, China).

Antibodies were obtained from different companies. The antibodies used in this study were as follows: Peroxidase AffiniPure Goat Anti-Mouse/Rabbit IgG (H+L) (33201ES60/33101ES60, Yeasen, Shanghai, China), anti-β-actin antibody (AC004, ABclonal, Wuhan, China), anti-Lamin A/C antibody (4777, Cell Signaling Technology, Danvers, USA), anti-α-Tubulin antibody (3873, Cell Signaling Technology), anti-acetylated Tubulin (T7451, Sigma-Aldrich), anti-Flag antibody (14793, Cell Signaling Technology), anti-GAPDH antibody (AC002, ABclonal), Alexa Fluor 488 AffiniPure Goat Anti-Mouse/Rabbit IgG (H+L) (33206ES60/33106ES60, Yeasen). Anti-NSUN7 antibody was generated through the N-terminal 1–188 amino acid residues of NSUN7 as an antigen to immunize rabbits.

Cell culture

HEK293T cells were purchased from the cell resource center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. The cells were authenticated by the provider using STR profiling. They were cultured in a 37°C incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (high glucose) (Corning, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and were routinely checked for mycoplasma via PCR. The cells used in this study were confirmed to be mycoplasma-free. The viable cell numbers were counted by trypan blue staining assays.

Animals

All animals were housed and treated according to the procedures approved by the Animal Ethics Committee of ShanghaiTech University, and all animal experiments were done in compliance with ethical guidelines and the approved protocols (Ethics No: 20231030001). The C57BL/6J mice were maintained in specific-pathogen-free conditions with a 12/12-h light/dark cycle, 22–26°C, 40–70% relative humidity, below 60 decibels, and with food and water ad libitum. CRISPR/Cas9 technology was used to generate Nsun7 KO mice by targeting exons 3 and 4 of the Nsun7 gene in the C57BL/6J background. The sgRNA sequences for generating Nsun7 KO mice were as follows:

sgRNA1: TTATGAATCCTGCATTCTTA
sgRNA2: GATCCAAGGTATGAACATGT

After zygote microinjection and embryo transplantation, genomic DNA from the tail tip of F0 mice was sequenced, and positive founders were selected for mating with wild-type C57BL/6J mice. Genomic DNA from F1 pups was sequenced to confirm germline transmission of the edited chromosome and absence of off-target hits. Genotyping of the Nsun7 KO mice was performed by sequencing cloned PCR products based upon the following primers:

Nsun7-identify-F: GCGTCCTGGGAATCAAACCA
Nsun7-identify-R: TGGGGACACCTTCAAGACATCA

Protein expression and purification

The coding sequence of Mus musculus NSUN7 (NM_001400914.1) was amplified from cDNA, which was obtained by RT-PCR from total RNA extracted from adult mouse testicular tissues. Then, the sequence was inserted into pET22b, along with a DNA sequence encoding a C-terminal His6-tag. Two separated truncated forms containing the genes encoding only 1–188 or 208–518 amino acid residues of NSUN7 were constructed according to the protocol provided with the KOD-plus mutagenesis kit. The constructs were verified by Sanger sequencing, and expressed in E. coli Rosetta (DE3). Recombinant NSUN7 or truncated NSUN7 proteins were purified by affinity chromatography on Ni-NTA Superflow resin. In short, the wet cells (~3g) were suspended in 15 mL of 10 mM imidazole in buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM DTT, and 10% (v/v) glycerol) and sonicated on ice. The crude extracts were centrifuged at 40,000 × g for 40 min to remove the debris and insoluble fractions. The supernatant was mixed gently with 0.5 mL Ni-NTA resin for 30 min at 4°C and then washed with 50 mL of 20 mM imidazole in buffer A to remove nonspecific binding proteins. Binding proteins were eluted in 8 mL of 250 mM imidazole in buffer A. The eluted protein was concentrated and purified by gel filtration on a Superdex 200 increase 10/300 GL column (GE Healthcare, USA). The protein concentrations were determined using UV absorbance at 280 nm, and the molar absorption coefficient was calculated according to the sequence of each protein81.

Isothermal titration calorimetry (ITC)

ITC measurements were performed at 25°C, using an ITC 200 MicroCalorimeter (MicroCal Inc., USA). Experiments included 20 injections of 2 μL of SAM (1 mM) into the sample cell containing 80 μM NSUN7 208–518 amino acid residues truncated protein. The protein and SAM were kept in the same buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM TCEP). SAM was titrated in the same buffer that was used as a control. Binding isotherms were fitted by nonlinear regression using Origin Software version 7.0 (MicroCal Inc.). The ITC data were fitted to a one-site binding model using Origin Software version 7.0 (MicroCal Inc.).

Western blotting

Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.45 μm or 0.22 μm PVDF membranes, and incubated with different antibodies. After blocking with 5% (w/v) non-fat dried milk, the membranes with targeted proteins were incubated with the corresponding primary antibodies overnight at 4°C. Membranes were then washed three times with PBST, and incubated with HRP-conjugated secondary antibody at room temperature (RT) for 30 min. After washing three times with PBST, the membranes were treated with the chemiluminescent substrates (EpiZyme, Shanghai, China), and imaging was performed using Amersham ImageQuant 800 (GE Healthcare).

Real-time quantitative PCR (RT-qPCR)

Total RNA was isolated using TRIzol according to the manufacturer’s instructions. The cDNA synthesis using total RNA as the template was performed with HiScript III RT SuperMix. RT-qPCR was performed using the relative standard curve method in QuantStudio 7 (Life Technology, USA) with ChamQ Universal SYBR QPCR Master Mix as the dsDNA fluorescence dye. The reactions were performed under the following conditions: 95°C for 2 min; 40 cycles of 95°C for 30 sec, 62°C for 20 sec, and 72°C for 30 sec; and a melting curve from 50°C to 95°C. The primers used for the corresponding mRNA in RT-qPCR assays are listed in Supplementary Data 1.

Quantitative analysis of tRNA modification using UPLC-MS/MS

The 200 ng of specific RNAs were hydrolyzed with 0.5 μL benzonase, 0.5 μL phosphodiesterase I, and 0.5 μL bacterial alkaline phosphatase in a 100 μL solution including 4 mM NH4OAc at 37°C overnight. After complete hydrolysis, the products were dissolved in acetonitrile, and subsequently applied to Ultra-Performance Liquid Chromatography-Mass Spectrometry/MS (UPLC-MS/MS). The nucleosides were separated on an Atlantis HILIC Silica column (2.1 × 150 mm, 3 μM) and then detected by a triple-quadruple mass spectrometer (AB Sciex QTRAP 6500+) in the positive ion multiple reaction-monitoring (MRM) mode. The nucleosides were quantified using the nucleoside-to-base ion mass transitions of 268.1 to 136.2 (A), 284.1 to 152.2 (G), 244.1 to 112.1 (C), 245.0 to 113.1 (U), and 258.1 to 126.1 (m5C). Quantification was performed by comparison to the standard curve obtained from pure nucleoside standards running in the same batch. The relative abundance of m5C was calculated as the ratio of m5C to the total A content.

Immunofluorescence microscopy

HEK293T cells were transfected with the pcDNA3.1-Nsun7-Flag plasmid. After transfection for 24 h, the cells were fixed in 4% paraformaldehyde (PFA) for 30 min and then permeabilized in 0.2% Triton X-100 for 5 min on ice. After washing with phosphate-buffered saline (PBS), fixed cells were blocked in PBS plus 0.1% Triton X-100 buffer containing 5% BSA and incubated with mouse anti-Flag antibodies at a 1:400 dilution overnight at 4°C. The cells were then immunolabeled with Alexa Fluor 488-conjugated goat anti-mouse IgG in PBS with a 1:1000 dilution for 2 h and the nuclear counterstain DAPI for 5 min at RT.

Mouse testes were fixed in 4% PFA overnight at 4 °C. After being washed in phosphate-buffered saline, the testes were perfused in 30% sucrose, and embedded in Tissue-Tek O.C.T. Compound. Tissue sections were cut at 10 μm and mounted on silanized slides. Then, the sections were permeated with enhanced immunostaining permeabilization buffer for 30 min at RT. After three washes with PBS, nonspecific binding was blocked by incubating the sections in blocking buffer for 30 min at RT. The sections were then incubated with rabbit anti-NSUN7 antibody at a 1:100 dilution overnight at 4°C. The cells were then immunolabeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG in PBS with a 1:1000 dilution for 2 h. All fluorescent images were taken and analyzed using an LSM880 confocal microscope (Zeiss, Germany).

Histology

Mouse testes and epididymides were dissected and fixed in Bouin’s solution (Leica, Germany) overnight at 4°C on a rotator, then the testes were cut into two pieces, after washing twice with phosphate-buffered saline, and dehydrated in a gradient series of different concentrations ethanol, followed by two xylene washes to remove ethanol, paraffin embedding, and sectioning at 5 μm thickness. For the histological analysis of testes or epididymides, sections were stained with hematoxylin and eosin or periodic acid-schiff after dewaxing and rehydration.

IVF

IVF was performed following a protocol from Jackson Laboratory (http://cryo.jax.org/ivf.html). In brief, ovarian stimulation was performed using standard gonadotropin-releasing hormone agonist downregulation and gonadotropin stimulation of the ovaries. Oocytes from 12-week-old mice were obtained using ultrasound-guided, transvaginal aspiration, and incubated in HTF medium (AiBei, Nanjing, China) supplemented with 15% heat-deactivated maternal sera for 4 h prior to sperm insemination. Insemination and culture were performed using standard “microdrop” techniques.

CASA

Sperm were harvested by dissecting cauda epididymides in 37°C pre-warmed Enriched Krebs-Ringer bicarbonate medium (EKRB medium; 120.1 mM NaCl, 4.8 mM KCl, 25.2 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.3 mM CaCl2, supplemented with 11.1 mM glucose, 2 mM glutamine, 1× essential amino acids, 1× nonessential amino acids, 100 μg/mL streptomycin, 100 U/mL penicillin). Then, sperm-containing medium was dropped into the calibrated slide for veterinary semen analysis (Leja).

TEM

Cauda epididymal spermatozoa were incubated in TYH medium to allow dispersion, collected into a 2.0-mL tube, and then fixed in 2.5% glutaraldehyde. After primary fixation, samples were washed with 0.1 M phosphate-buffered saline, and postfixed with 1% osmium tetroxide. Dehydration was carried out in a gradient series of ethanol, and infiltrated with acetone. Ultimately, samples were embedded in Epon 812. Ultrathin sections with 70 nm thickness were prepared by ultramicrotome (Leica UC7, Leica Biosystems, Germany), and counterstained with uranyl acetate and lead citrate. Images were taken with a Zeiss Gemini SEM 460 Microscope (Germany).

Isolation of mouse testicular haploid spermatids

We isolated haploid spermatids from mouse testes with a simple protocol82 based on the underlying principle of STA-PUT velocity sedimentation54,55. Testes were decapsulated, and gently teased apart with forceps to obtain the seminiferous tubules. Then, the seminiferous tubules were incubated with 1 mg/mL collagenase I in 1× Krebs at 37°C in a water bath for 10–15 min. The sedimented tubules were further digested with 0.05% trypsin containing DNase I, and gently pipetted up and down for 5–15 min before adding DMEM containing 10% FBS. Cells were pelleted by centrifugation at 500 × g for 5 min, and then resuspended in 1× Krebs, and filtered with a 40 μm cell strainer to obtain a monodisperse suspension. The single-cell suspension was loaded into a 50 mL tube, which contained a discontinuous gradient from 5% to 1% BSA in 1× Krebs from bottom to top with 5 mL per layer, and then allowed to sediment by gravity for 1.5–2 h at 4°C. The haploid spermatids were carefully collected in ~1 mL fractions in separate 1.5 mL tubes for a total of 4–5 tubes, starting from the top of the BSA gradient. Cell purity was checked under a DIC light microscope based on distinctive diameters and morphological features, and validated under an immunofluorescence microscope based on staining patterns of DAPI.

Separate different types of RNA

Total RNA from testes or haploid spermatids was extracted using TRIzol, and precipitated overnight with ethanol at −20°C. mRNA can be captured from total RNA using beads conjugated with oligo-dT (Vazyme, China). For the separation of RNA larger than 200 nt or smaller than 200 nt, RNA Clean & Concentrator-5 (Zymo, USA) was utilized according to the manufacturer’s protocol. In order to obtain 18S and 28S rRNA, total RNA was separated by agarose gel electrophoresis, and recovered with Gel RNA Recovery kit (Zymo, USA) according to the manufacturer’s protocol.

UBS-seq library construction and sequencing

Bisulfite treatment was performed using UBS-2 buffer as described in ref. 57. In brief, 45 µL of UBS-2 reagent was preheated to 98°C for 5 min in a PCR instrument. Subsequently, approximately 0.1–1 μg of RNA dissolved in 5 µL of water was added to the reagent and thoroughly mixed. The mixture was then incubated at 98°C for 9 min with the lid temperature set to 105°C. After cooling to RT, desulphonation was performed according to the manufacturer’s protocol using the EZ RNA Methylation Kit (Zymo, USA). The quantity of converted RNA was measured using a Qubit.

For mRNA UBS-seq library preparation, the Stranded mRNA-seq LibPrep Module for Illumina (ABclonal, China) was used. For large RNA UBS-seq library preparation, the converted RNA samples were fragmented into small RNAs by heat treatment in the presence of 5 mM MgCl2, and then subjected to end-repair using T4 Polynucleotide Kinase (PNK). Finally, the libraries were constructed using the VAHTS Small RNA Library Prep Kit for Illumina V2 (Vazyme, China). For small RNA UBS-seq library preparation, the converted RNA samples underwent end-repair with T4 PNK and subsequently processed into sequencing libraries using VAHTS Small RNA Library Prep Kit for Illumina V2 (Vazyme, China). Libraries were all sequenced at Majorbio (China). Briefly, all libraries underwent quality control assessment using an Agilent TapeStation. Those libraries that passed quality control were sequenced on the NovaSeq X plus (Illumina, USA), generating paired-end 150 bp reads. Each group had three biological replicates. Comparative analysis of site-specific RNA m5C level between WT and Nsun7−/− group was performed two-sided Fisher’s exact test to calculate p value. Differences were considered to be statistically significant when p value < 0.05.

UBS-seq data analysis

The UBS-seq data were analyzed using the previously described pipeline36, with some modifications. In brief, we performed trimming of adapter sequences and low-quality reads with cutadapt83 and trimmomatic84. Paired reads and unpaired reads were then mapped separately to the C–T, G–A transformed mouse genome (GRCm39) with HISAT285. Unmapped and multiple-mapped reads were further mapped to a C-to-T converted transcriptome by Bowtie286. The transcript coordinates were adapted to match the genomic locations based on the mouse Ensembl GTF annotation. We analyzed the paired reads and unpaired reads, and merged them into the total BAM files. For large RNA UBS-seq data analysis, in addition to GRCm39, alignment references included the BK000964.3 and other rRNA variants from the SILVA database (https://www.arb-silva.de/). For small RNA UBS-seq data analysis, transcript coordinates were genomically aligned using a composite annotation system incorporating the annotations from Ensemble, RNAcentral87, Genomic tRNA database88, piRbase89, and miRbase90. We set the criteria for potential m5C modification site calling to have a coverage of C+T above 15 and a base quality score above 30. We used a binomial model to assess the p value for each potential m5C site.

Proteomic profiling by DIA-MS

Proteins were extracted from wild-type or Nsun7 KO mouse testis samples using SDT lysis buffer (4% SDS, 100 mM DTT, 100 mM Tris-HCl, pH 8.0). Samples were boiled and further ultrasonicated. Undissolved cellular debris was removed by centrifugation, and the supernatant was collected and quantified with a BCA Protein Assay Kit (Beyotime, China). Protein digestion was performed with the FASP method described in ref. 91. LC-MS/MS was performed on an Orbitrap Astral mass spectrometer coupled with a Vanquish Neo UPLC system (Thermo Fisher Scientific, USA). The DIA method consisted of a survey scan from 380-980 m/z at resolution 240,000 with AGC target of 500% and 5 ms injection time. The DIA MS/MS scans were acquired by Astral from 150–2,000 m/z with a 2 m/z isolation window and with AGC target of 500% and 3 ms injection time. Normalized collision energy was set to 25, and cycle time was 0.6 sec. The spectra of full MS scan and DIA scan were recorded in profile and centroid type, respectively. p values were determined by a two-tailed unpaired Student’s t-test. Proteins with log2(fold change) ≥ 1.0 (upregulation) or ≤ −1.0 (downregulation), accompanied by a p value < 0.05, were considered significantly differentially expressed.

Preparation of single-cell suspensions

The fresh wild-type or Nsun7 KO 12-week-old mouse testis samples were minced, and enzymatically digested using the MACS Tumor Dissociation Kit (Miltenyi, China) for 30 min with agitation, according to the manufacturer’s instructions. The dissociated cells were subsequently passed through a 70 μm and 40 μm cell-strainer and centrifuged at 300 g for 10 min. The pelleted cells were then suspended in red blood cell lysis buffer (Tiangen, China), and washed with 0.04% BSA in DPBS. Finally, the cell concentration was adjusted to 1000 cells/μL (viability ≥ 80%) in all samples.

Single-cell RNA library construction, sequencing, and data processing

Single-cell RNA-seq libraries were prepared with DNBelab C Series High-throughput Single-Cell RNA Library (MGI, China) according to the manufacturer’s instructions. Libraries were purified and quantified using the Qubit ssDNA kit (Thermo Fisher Scientific, USA) and Qsep100 (Bioptic, China), and subsequently sequenced on DNBelab C4/DNBelab TaiM4 Series Single-Cell Library Prep Set (MGI, China). PISA was used to calculate the gene expression of cells and create a gene × cell matrix for each library.

The “Seurat” R package was then used to perform single-cell RNA-seq analyses. Count matrices were merged using Seurat version 5.2.192, and a percentage of mitochondrial genes was calculated. Cells containing fewer than 300 identified genes or more than 20% of reads arising from mitochondrial genes were removed. For normalization and feature selection, the Seurat functions “NormalizeData”, “FindVariableFeatures” (n.features = 2000), and “ScaleData” were used. Two-dimensional representations were generated by using the outputs from PCA (Principal Component Analysis) and Harmony as inputs to the “RunUMAP” functions. Differential expression gene analysis was performed using the nonparametric two-sided Wilcoxon rank-sum test. Cell types were allocated to each cluster using the abundance of known marker genes as described previously61. p values of cell clusters between the WT and Nsun7−/− group were calculated using the nonparametric two-sided Wilcoxon rank-sum test. Adjusted p values were used with the Bonferroni correction. Prior to differential gene analysis across cell clusters, we filtered for genes expressed in > 10% of the WT group (pct.1 > 0.1). From the filtered genes, only those with adjusted p value < 0.05, average log2(fold change) ≥ 1.5 (upregulation) or ≤ −1.5 (downregulation) were considered significantly differentially expressed.

Statistics and reproducibility

Statistical analyses and data visualization were performed using GraphPad Prism version 10.4.1 and R version 4.3.0. Image visualization was performed using ImageJ (version 1.54). Error bars represent mean ± SD for three independent experiments. Comparisons between two groups were analyzed using a two-tailed unpaired Student’s t-test or a two-sided Fisher’s exact test, as indicated. For more groups, statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance was defined as p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their manuscript “NSUN7 is an RNA:m5C catalytically inactive protein that regulates sperm flagellum assembly” Li et al., using RNA mass spectrometry and bisulfite sequencing, show that knockout of Nsun7 does not affect m5C modification levels, suggesting that the protein is a catalytically inactive member of the NSUN family. Moreover, the absence of Nsun7 leads to severe flagellar microtubule defects, which impair sperm motility and are critical for male fertility.\nThe manuscript is well-written and logically structured. I particularly appreciate that the authors coherently presented the negative results and the absence of effects on m5C methylation related to NSun7.\nHowever, the following major points should be addressed before the manuscript can be considered for publication.\n1) Pag 12 row 13: The authors cannot rule out the possibility that tissues not included in their analysis, such as ovary or from the female reproductive tract, also express the protein. Notably, epithelial cells of the fallopian tube possess complex motile cilia that facilitate egg transport from the ovary to the uterus. Investigating NSun7 expression in these cells could help clarify its tissue-specific distribution and functional relevance. In any case, I recommend revising the paragraph to reflect this consideration and avoid using the term “exclusively”.\n2) Fig 3: It should be clearly excluded that also mitochondrial RNA is not modified by NSUN7. I believe the authors could reanalyze their bisulfite sequencing data, highlighting or plotting all sequenced mitochondrial RNAs.\n3) Please include a positive control in Figure 4G. Furthermore, the observation that the NSUN domain of human NSUN7 fails to bind SAM due to the substitution of Asp with Leu in Motif IV is very intriguing and supports the lack of methylation activity, as shown by the MS and bisulfite data. The authors should test their hypothesis by reverting the Leu back to Asp in the NSUN domain of human or mouse NSUN7, potentially restoring the domain’s SAM-binding capacity.\n4) Figure 7: The observed change in gene expression cannot be attributed solely to a structural function of NSUN7. The mechanisms underlying its regulatory role remain unclear based on the presented results. The authors should provide further clarification on this point.\nMinor points:\nFigure 2b: The x axis categories are not correctly labeled.", "reviewer_2": "Dear Editor of Nature Communication,\nWe do thank you very much for inviting us to co-review the manuscript \"NSUN7 is an RNA:m5C catalytically inactive protein that regulates sperm flagellum assembly\" by Professor Liu and colleagues. The following comments are co-written by Dr Shi Shuo and me.\nMajor comments:\nThis study presents compelling evidence that NSUN7 is an RNA:m5C catalytically inactive protein, in contrast to other NSUN family members, and that knockout of NSUN7 leads to significant defects in the axoneme structure and mispositioning of longitudinal columns in elongated spermatids. This, coupled with the observed downregulation of cilium organization-associated genes in the same cell type, clearly establishes a critical non-catalytic regulatory role for NSUN7 in sperm flagellar cytoskeleton organization. The finding that NSUN7 specifically localizes to the sperm flagella is particularly important. Overall, this is a very good research article, the study aim is clear, and the experiments are well designed.\nHowever, this precise localization raises an intriguing mechanistic question that I believe warrants further discussion in the manuscript: How does the loss of NSUN7’s ASM binding and RNA m5C methylation catalytic capacity, a protein residing on the flagella (a structure quite distant from the nucleus), lead to reduced mRNA levels for these specific genes?\nThe authors have appropriately discussed this point (and mentioned their attempts to find interacting partners, hampered by antibody issues). It’s a fascinating puzzle. Could NSUN7 potentially have a direct role in binding and regulating the stability of its target mRNAs? The recent suggestion that related proteins might have RNA-binding activity (Guseva E, et al., RNA 2025) makes this an interesting possibility, though far from proven. Alternatively, and perhaps more likely given its flagellar location, is the downregulation an indirect consequence? The severe structural defects in the axoneme and longitudinal columns might disrupt cellular processes needed for the proper storage, localization, or stability of these specific mRNAs. Maybe it triggers a stress response affecting mRNA levels broadly or specifically.\nWhile the exact mechanism linking the flagellar defect to reduced mRNA expression remains an open question, it doesn’t diminish the core findings regarding NSUN7’s essential non-catalytic regulatory role in flagellar structure assembly. Figuring out how these two phenotypes are connected is a really interesting direction for future work. Approaches like looking at mRNA stability, localization, or exploring signaling pathways, especially cytoskeleton dynamics of microtubule-organizing centers (MTOC) during spermiogenesis could shed light on this in follow-up studies. Clarifying this point would certainly deepen our understanding, but the current results on NSUN7’s structural role are already significant and well-supported.\nMinor Comment:\n• In the Abstract, the abbreviation SAM is used upon its first mention. For clarity, it should be defined as S-adenosylmethionine (SAM).\n• Page 3, Line 37: in the instruction section, there is a typo of “In Sum” needed to be corrected.\n• Page 14, Line 37-38: In describing the structure-mechanism relationship of NSUN7 relevant to RNA:m5C methylation process, with comparing to other NSUN members, since NSUN7 does not naturally possess SAM-binding capability, as the authors reported, the phase “loss of SAM binding capability” seems confusing to the readers by assuming that the lack of such binding capability can be due to artificially induced dysfunction such as by knockout, and thus the reviewer suggests using other term, like “devoid’ or “lack” of “SAM binding capability” to avoid confusion.\n• Given that NSUN7 is essential for sperm flagellum longitudinal columns positioning and cilium organization, and that it can also enter the nuclues (albeit at low levels) revealed in HEK293 cells, and its expression abundance in all late stages of sperm morphological transformation, its mechanistic role in microtubule cytoskeleton dynamics during late-stage spermatogenesis, such as involvement of centrosome, a type of MTOC, in spermiogenesis and spermatozoan mRNA storage also warrants further discussion. In this regard, the expression abundance of the DEGs, although their numbers are lower in spermatocytes and round spermatocyte than in elongated spermatocytes, these DEGs should also be considered, as this could indicate their essential role as non-enzymatic proteins. As NSUN7 colocalizes with acetylated tubulin, further in-depth interpretation of the single-cell omics may provide some hints on what potential microtubule proteins or pathway involving NSUN7, which is also worth mentioning in the discussion.", "reviewer_3": "This manuscript presents a compelling and well-structured study that addresses a long-standing question regarding the function of NSUN7, a poorly characterized member of the NSUN family of RNA methyltransferases. The authors provide strong evidence that NSUN7 lacks canonical catalytic activity due to key mutations in its SAM-binding domain, distinguishing it from other NSUN proteins. They convincingly show that NSUN7 is enriched in elongated spermatids, and that its loss leads to defects in sperm motility and axonemal structure, accompanied by downregulation of cilium-related transcripts.\nThese findings are significant and novel, as they establish NSUN7 as a non-catalytic regulator with a potential structural or scaffolding role during spermatogenesis. However, the underlying molecular mechanism remains incompletely explored, particularly in terms of RNA and protein interactions. Addressing the key questions below will be essential to fully define NSUN7’s biological role and support its proposed function in male fertility.\nTo substantiate the novel model proposed—where NSUN7 functions in a non-enzymatic, regulatory capacity—the manuscript would benefit greatly from mechanistic data that clarify NSUN7’s molecular interactions and subcellular dynamics. The following experiments and clarifications will strengthen the biological conclusions and are likely essential for publication in a high-impact venue.\nMajor Comments:\n1. Is NSUN7's localization RNA-dependent in human and mouse samples? The authors should assess whether RNase treatment alters NSUN7 localization, using immunostaining or fractionation. This would help determine if RNA binding anchors NSUN7 to specific compartments. And whether NSUN7 localization is conserved in mouse and human.\n2. Is NSUN7 expression paterns conserved in human tissues?\n3. Does NSUN7 retain RNA-binding ability despite lacking methyltransferase activity? RIP or iCLIP using epitope-tagged NSUN7 in spermatids or other cell types would clarify its RNA targets and support a scaffolding or post-transcriptional regulatory role.\n4. Determining NSUN7’s protein interactors, especially in relation to microtubule proteins at the flagella, is critical. Co-immunoprecipitation followed by mass spectrometry using tagged NSUN7 could uncover partners relevant to its structural role in sperm motility.\n5. Since human infertility has been linked to point mutations in NSUN7, the authors should discuss or experimentally test whether such mutations disrupt NSUN7’s interaction with key structural proteins, contributing to defective sperm motility.\nMinor Comments:\nFigure 2B: Labels for 18S/WT and 18S/KO are missing in the bar chart and should be added for clarity.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to comments from reviewers:\nReviewer #1 (Remarks to the Author):\nIn their manuscript “NSUN7 is an RNA:m5C catalytically inactive protein that\nregulates sperm flagellum assembly” Li et al., using RNA mass spectrometry and\nbisulfite sequencing, show that knockout of Nsun7 does not affect m5C modification\nlevels, suggesting that the protein is a catalytically inactive member of the NSUN\nfamily. Moreover, the absence of Nsun7 leads to severe flagellar microtubule defects,\nwhich impair sperm motility and are critical for male fertility.\nThe manuscript is well-written and logically structured. I particularly appreciate that\nthe authors coherently presented the negative results and the absence of effects on m5C\nmethylation related to NSun7.\nResponse: We sincerely thank the reviewer for the insightful evaluations and the\nsupportive comments about our study. We have revised the manuscript carefully\naccording to these valuable suggestions and added or updated the corresponding\nFigures, Supplementary Figures and Tables.\nHowever, the following major points should be addressed before the manuscript can be\nconsidered for publication.\n1) Pag 12 row 13: The authors cannot rule out the possibility that tissues not included\nin their analysis, such as ovary or from the female reproductive tract, also express the\nprotein. Notably, epithelial cells of the fallopian tube possess complex motile cilia that\nfacilitate egg transport from the ovary to the uterus. Investigating NSun7 expression in\nthese cells could help clarify its tissue-specific distribution and functional relevance. In\nany case, I recommend revising the paragraph to reflect this consideration and avoid\nusing the term “exclusively”.\nResponse: We sincerely thank the reviewer for this thoughtful comment. We fully\nagree that it is necessary to consider the possibility that NSUN7 may also be expressed\nin female reproductive tissues, such as the ovary and fallopian tube, where epithelial\ncells possess motile cilia that play key roles in oocyte transport. To address this concern,\nwe carefully examined NSUN7 expression in a range of female reproductive tissues.\nSpecifically, we isolated ovary, fallopian tube, uterus, and vaginal tissues from adult\nfemale mice and analyzed NSUN7 protein levels using Western blotting with testis\nserving as a positive control. We found that NSUN7 protein was too low to be detected\nin these female reproductive tissues as shown in the revised Figure S2, as shown below.\n1 / 18\nBecause the female mouse reproductive tissues comprise multiple cell populations,\nit remains possible that Nsun7 is expressed in specific cell types. To further explore this\npoint, we analyzed published single-cell RNA sequencing datasets of mouse ovarian\nsurfaces and fallopian tubes (Qin G, et al., iScience, 2022) to examine Nsun7 expression,\nparticularly in the fallopian tube epithelial ciliated cells.\nUMAP analysis of this dataset revealed five cell clusters (fallopian tube epithelial\nsecretory cells, fallopian tube epithelial ciliated cells, ovarian surface epithelial cells,\nfallopian tube and ovarian stromal cells, hemocyte cells) (Response Figure 1A). The\nresults showed that Nsun7 mRNA was mainly enriched in ovarian and fallopian tube\nstromal cells, followed by fallopian tube epithelial ciliated cells (Response Figure 1B).\nIn different cell clusters of the fallopian tubes and ovaries, we observed that only a\nsmall number of cells contained Nsun7 mRNA (Response Figure 1B).\nWe integrated these single-cell sequencing data and ours to provide a reference for\nNsun7 expression levels across different cell types. However, considering sequencing\ndepth and batch effects, these results should be interpreted as reference information\nonly. The average expression levels were calculated using the integrated log-\nnormalized values from the integrated assay. We showed that compared with\nspermatocyte, elongated spermatids and round spermatids in the testis, Nsun7 is\nexpressed at low levels across different cell types including fallopian tube epithelial\nciliated cells in the fallopian tube and ovary (Response Figure 1C), which is consistent\nwith low NSUN7 protein levels detected in female reproductive tissues. In short, our\nresults demonstrate that NSUN7 exhibits low protein levels in female reproductive\ntissues. Accordingly, we have now revised the relevant paragraph on current Page 12,\nLine 13 and removed the term “exclusively” as suggested.\n2 / 18\nResponse Figure 1\n(A) UMAP plot of single cells from fallopian tube and ovary surface of adult female mice, revealing\nfive clusters of cell types.\n(B) Violin plot showing Nsun7 expression levels across five clusters of fallopian tube and ovarian\ncell types.\n(C) The average expression level of Nsun7 in different cell types was calculated using integrated\nlog-normalized values.\n2) Fig 3: It should be clearly excluded that also mitochondrial RNA is not modified by\nNSUN7. I believe the authors could reanalyze their bisulfite sequencing data,\nhighlighting or plotting all sequenced mitochondrial RNAs.\nResponse: Thanks for the insightful suggestion. We fully agree that it is important to\nclearly exclude the possibility that mitochondrial RNAs are modified by NSUN7. We\nhave carefully reanalyzed our UBS-seq (ultrafast bisulfite sequencing) datasets to\nspecifically assess m5C modification patterns in mitochondrial RNAs.\nFor mitochondrial RNAs, we identified a total of nine m5C modification sites, located\non 12S rRNA and six tRNAs. Knockout of Nsun7 did not affect the m5C modification\nsites or their modification levels in mitochondrial RNAs (Figure 3J; Table S5). We\ndetected m5C modification at position 842 on 12S rRNA (position 911 in the\nmitochondrial genome), which remained unchanged in Nsun7-/- mouse testes compared\nto WT controls (Figure 3K). Additionally, we detected eight m5C modification sites\nacross six mitochondrial tRNAs (mt-tRNA-Ser2, mt-tRNA-His, mt-tRNA-Leu1, mt-\ntRNA-Leu2, mt-tRNA-Met, mt-tRNA-Pro), exhibiting distinct modification levels. We\nfound that knockout of Nsun7 had no impact on m5C modification levels in these\nmitochondrial tRNAs (Figure 3L).\nTherefore, we have now plotted all sequenced mitochondrial RNAs containing m5C\nmodifications, which are now presented in the revised Figures 3J-L (as shown below)\n3 / 18\nand summarized in Table S5, with descriptions on current Page 14, Lines 31-39.\n3) Please include a positive control in Figure 4G. Furthermore, the observation that the\nNSUN domain of human NSUN7 fails to bind SAM due to the substitution of Asp with\nLeu in Motif IV is very intriguing and supports the lack of methylation activity, as\nshown by the MS and bisulfite data. The authors should test their hypothesis by\nreverting the Leu back to Asp in the NSUN domain of human or mouse NSUN7,\npotentially restoring the domain’s SAM-binding capacity.\nResponse: We sincerely thank the reviewer for this insightful suggestion. We fully\nagree that experimentally testing whether the Leu-to-Asp substitution in Motif IV\nrestores SAM-binding capacity would provide important mechanistic validation of our\nhypothesis.\nBuilding on the reviewer’s valuable recommendation, we generated a NSUN domain\nmutant of mouse NSUN7, in which a Leu379-to-Asp substitution was introduced in\nMotif IV. Both wild-type and L379D mutant NSUN domains of mouse NSUN7 were\npurified to high purity (now Figure S4A), and their SAM-binding capacities were\nexamined using isothermal titration calorimetry (ITC). Consistent with our original\nresults, the wild-type NSUN domain (mNSUN7 WT) cannot bind to SAM (now\nFigures S4B, C). We then performed three independent gradual titrations of SAM into\nthe L379D mutant NSUN (mNSUN7 L379D) domain, all of which yielded consistent\nresults. The representative image is shown as Figure S4D. During the initial injections,\ndistinct exothermic signals were observed. Subsequently, at the 5th and 8th injections,\ndistinct exothermic and endothermic peaks were detected, probably resulting from\nSAM-induced conformational changes in the protein during titration. In the subsequent\nSAM titrations, the exothermic signals gradually weakened and eventually stabilized.\nThese results suggest that the L379D mutant NSUN domain is capable of binding SAM.\nWe have now added these results and descriptions on current Page 16, Lines 1-13.\n4 / 18\n4) Figure 7: The observed change in gene expression cannot be attributed solely to a\nstructural function of NSUN7. The mechanisms underlying its regulatory role remain\nunclear based on the presented results. The authors should provide further clarification\non this point.\nResponse: We sincerely appreciate the reviewer’s insightful comment highlighting that\nthe observed change in gene expression cannot be attributed solely to a structural\nfunction of NSUN7. During the preparation of this manuscript, Guseva E, et al.\ngenerated HA-tagged NSUN7 mice, and used anti-HA antibody to perform CLIP in\nmouse testis and found that NSUN7 binds specific mRNAs, with one-third of these\nmRNAs being specific to elongated spermatids, supporting its potential RNA-binding\ncapability. Interestingly, when we treated testis lysate with RNase A, the endogenous\nNSUN7 protein levels decreased. Treatment with RNase A also led to a decrease in the\nlevels of overexpressed human NSUN7 protein in cultured cells. (Now Figures S9A,\nB). The corresponding results are now described in the revised manuscript (Page 19,\nLines 27-33). Together, these results suggest that NSUN7 possesses RNA-binding\ncapability, and the association of NSUN7 with RNA contributes to maintaining its\nprotein stability.\nNevertheless, it remains uncertain whether the reduced expression of cilium\n5 / 18\norganization-associated genes results from this potential RNA-related role or from the\nsevere structural defects observed in the axoneme and longitudinal columns. Therefore,\ndistinguishing cause from effect remains challenging and awaits future studies in\nemploying advanced single-molecule and high-resolution RNA-protein interaction\ndetection approaches. We have clarified this point in the Discussion section (Page 22,\nLines 18-30).\nMinor points:\nFigure 2b: The x axis categories are not correctly labeled.\nResponse: We sincerely thank the reviewer for the careful reading and we apologize\nfor the mistake. We have corrected the x axis in Figure 2B.\nReviewer #2 (Remarks to the Author):\nMajor comments:\nThis study presents compelling evidence that NSUN7 is an RNA:m5C catalytically\ninactive protein, in contrast to other NSUN family members, and that knockout of\nNSUN7 leads to significant defects in the axoneme structure and mispositioning of\nlongitudinal columns in elongated spermatids. This, coupled with the observed\ndownregulation of cilium organization-associated genes in the same cell type, clearly\nestablishes a critical non-catalytic regulatory role for NSUN7 in sperm flagellar\ncytoskeleton organization. The finding that NSUN7 specifically localizes to the sperm\nflagella is particularly important. Overall, this is a very good research article, the study\naim is clear, and the experiments are well designed.\nResponse: We truly appreciate the reviewers’ recognition and positive feedback of our\nwork.\nHowever, this precise localization raises an intriguing mechanistic question that I\nbelieve warrants further discussion in the manuscript: How does the loss of NSUN7’s\nASM binding and RNA m5C methylation catalytic capacity, a protein residing on the\nflagella (a structure quite distant from the nucleus), lead to reduced mRNA levels for\nthese specific genes?\nResponse: We sincerely thank reviewers’ insightful question. We fully agree that\nunderstanding how a catalytically inactive protein localized to the flagellum could\ninfluence mRNA expression is a key and thought-provoking aspect of our work.\nThe transformation from round to elongated spermatids, known as spermiogenesis,\nis a complex process involving extensive cellular remodeling. Elongated spermatids are\n6 / 18\nhighly compact and specialized cells characterized by tightly condensed chromatin and\nminimal cytoplasm. We detected NSUN7 localization in the flagellar region of\nelongated spermatids, consistent with a recent study (Guseva E, et al., RNA, 2025) using\nan HA-tagged NSUN7 mouse model. It is noteworthy that our results show that Nsun7\nis highly expressed at postnatal day 28, which corresponds well to the developmental\nstage at which elongated spermatids first appear in mouse testes.\nThus, it seems possible that NSUN7 may play a role during spermiogenesis, leading\nto the reduced mRNA levels of cilium organization-associated genes observed in\nelongated spermatids. However, due to current limitations in antibody specificity and\ndetection sensitivity, it remains challenging to precisely determine the subcellular\nlocalization of NSUN7 during spermiogenesis, and to define how NSUN7 regulates\nthese cilium organization-related mRNAs in elongated spermatids. Future studies may\nrequire improving the specificity and sensitivity of the NSUN7 antibody to perform\nimmuno-electron microscopy or single-molecule localization microscopy to study its\nprecise localization, as well as employing single-molecule techniques to investigate the\neffect of NSUN7 on the expression of cilium organization-associated genes. We have\nclarified this point in the Discussion section (Page 22, Lines 10-17).\nThe authors have appropriately discussed this point (and mentioned their attempts to\nfind interacting partners, hampered by antibody issues). It’s a fascinating puzzle. Could\nNSUN7 potentially have a direct role in binding and regulating the stability of its target\nmRNAs? The recent suggestion that related proteins might have RNA-binding activity\n(Guseva E, et al., RNA 2025) makes this an interesting possibility, though far from\nproven. Alternatively, and perhaps more likely given its flagellar location, is the\ndownregulation an indirect consequence? The severe structural defects in the axoneme\nand longitudinal columns might disrupt cellular processes needed for the proper storage,\nlocalization, or stability of these specific mRNAs. Maybe it triggers a stress response\naffecting mRNA levels broadly or specifically.\nResponse: We sincerely thank reviewers for this thoughtful comment. A recent study\n(Guseva E, et al., RNA, 2025) used an HA-tagged NSUN7 mouse as mouse model, and\nusing HA antibody to perform CLIP in mouse testis and found that NSUN7 binds\nspecific mRNAs, and one-third of these mRNAs being specific to elongated spermatids,\nsupporting its potential RNA-binding capability. Interestingly, when we treated testis\n treated testis\nlysates with RNase A, the endogenous NSUN7 protein levels decreased (Now Figure\nS9A). A decrease in the levels of overexpressed human NSUN7 protein was also\nobserved in cells following RNase A treatment (Now Figure S9B). These results are\nnow described in the revised manuscript (Page 19, Lines 27-33). Together, these results\nsuggest that NSUN7 possesses RNA-binding capability, and the association of NSUN7\nwith RNA contributes to maintaining its protein stability.\n7 / 18\nNevertheless, we cannot assess whether the decrease in the expression of cilium\norganization-associated genes is due to the potential regulatory role of NSUN7 as an\nRNA-binding protein or as a result of severe structural defects in the axoneme and\nlongitudinal columns. The decrease in gene expression levels and the structural defects\ncaused by Nsun7 deficiency require further investigation. Future work employing\nadvanced single-molecule and high-resolution RNA-protein interaction detection\napproaches will be essential to determine whether NSUN7 possesses direct RNA-\nbinding affinity, and whether the observed decrease in cilium organization-associated\nmRNA levels is a cause of defective sperm flagellum assembly due to Nsun7 deficiency,\nor a consequence of structural abnormalities. We have integrated this interpretation into\nthe revised Discussion (Page 22, Lines 18-30).\nWhile the exact mechanism linking the flagellar defect to reduced mRNA expression\nremains an open question, it doesn’t diminish the core findings regarding NSUN7’s\nessential non-catalytic regulatory role in flagellar structure assembly. Figuring out how\nthese two phenotypes are connected is a really interesting direction for future work.\nApproaches like looking at mRNA stability, localization, or exploring signaling\npathways, especially cytoskeleton dynamics of microtubule-organizing centers (MTOC)\nduring spermiogenesis could shed light on this in follow-up studies. Clarifying this\npoint would certainly deepen our understanding, but the current results on NSUN7’s\nstructural role are already significant and well-supported.\nResponse: We sincerely appreciate the reviewers’ thoughtful and encouraging\nevaluation, as well as the insightful suggestions for future studies. Recently, we\nconducted immunoprecipitation followed by mass spectrometry to detect NSUN7-\ninteracting proteins under treatment with or without RNase A. Flag-tagged mouse\nNSUN7 was first overexpressed in cells. Then, the cells were combined with mouse\ntestis and lysed together, and Flag-IP MS assays were used to pull-down potential\nNSUN7-interacting partners (Response Figure 4A). The MS results showed a\nsignificant increase in β-tubulin and α-tubulin proteins in NSUN7 IP samples compared\nto the control IP group (Response Figures 4B-D). However, after RNase A treatment,\nthe tubulin signal intensity in NSUN7 IP samples became comparable to that in the\ncontrol IP samples (Response Figures 4B-D), indicating that NSUN7 may interact with\ntubulin proteins in the presence of RNA.\nHowever, when validating the IP samples, we observed a high background of tubulin\nbinding in both control and NSUN7 IP groups. It is difficult to conclude that NSUN7\nmay interact with tubulin proteins in the presence of RNA. Notably, NSUN7 protein\nlevels showed an obvious decrease after RNase A treatment. After RNase A treatment,\nNSUN7 appears not to bind tubulin, but tubulin exhibited increased non-specific\nbinding to the IP beads under these conditions (Response Figure 4E). Thus, other\n8 / 18\npossibilities may exist: (i) The reduced NSUN7 protein levels after RNase A treatment\nmay weaken its interaction with tubulin; (ii) increased non-specific binding of tubulin\nmay mask the interaction signal. Together, due to the limitation of this detection method,\nwe cannot conclude that NSUN7 interacts with tubulin, nor identify which specific\ntubulin isoforms it may interact with. The potential interacting partners of NSUN7,\nthe role of RNA in this process, and NSUN7-involved signaling pathway still await further\nvalidation using high-sensitivity and high-resolution approaches.\nWe fully agree that using high-precision detection method to assess mRNA stability,\nlocalization, or exploring signaling pathways, especially cytoskeleton dynamics of\nmicrotubule-organizing centers (MTOC) during spermiogenesis could provide valuable\ninsights in future studies. We have now incorporated this part in the revised Discussion\n(Page 22, Lines 4-9)\nMinor Comment:\n• In the Abstract, the abbreviation SAM is used upon its first mention. For clarity, it\nshould be defined as S-adenosylmethionine (SAM).\nResponse: Thanks. We have defined S-adenosylmethionine (SAM) in the Abstract to\nfacilitate readers’ comprehension.\n• Page 3, Line 37: in the instruction section, there is a typo of “In Sum” needed to be\ncorrected.\nResponse: Thanks. We have corrected “In Sum” to “In summary” on Page 3, Line 37.\n• Page 14, Line 37-38: In describing the structure-mechanism relationship of NSUN7\nrelevant to RNA:m5C methylation process, with comparing to other NSUN members,\nsince NSUN7 does not naturally possess SAM-binding capability, as the authors\nreported, the phase “loss of SAM binding capability” seems confusing to the readers by\nassuming that the lack of such binding capability can be due to artificially induced\ndysfunction such as by knockout, and thus the reviewer suggests using other term, like\n“devoid’ or “lack” of “SAM binding capability” to avoid confusion.\nResponse: Thanks for your helpful suggestion. The term \"loss or lose\" has been revised\nto \"lack\" throughout the manuscript, as seen in the Abstract and Page 15, Line 7.\n• Given that NSUN7 is essential for sperm flagellum longitudinal columns positioning\nand cilium organization, and that it can also enter the nuclues (albeit at low levels)\nrevealed in HEK293 cells, and its expression abundance in all late stages of sperm\nmorphological transformation, its mechanistic role in microtubule cytoskeleton\ndynamics during late-stage spermatogenesis, such as involvement of centrosome, a type\nof MTOC, in spermiogenesis and spermatozoan mRNA storage also warrants further\n9 / 18\ndiscussion. In this regard, the expression abundance of the DEGs, although their\nnumbers are lower in spermatocytes and round spermatocyte than in elongated\nspermatocytes, these DEGs should also be considered, as this could indicate their\nessential role as non-enzymatic proteins. As NSUN7 colocalizes with acetylated tubulin,\nfurther in-depth interpretation of the single-cell omics may provide some hints on what\npotential microtubule proteins or pathway involving NSUN7, which is also worth\nmentioning in the discussion.\nResponse: We are very grateful to the reviewers for the insightful and integrative\ncomments. As you pointed out, our immunofluorescence and expression analyses show\nthat NSUN7 is highly enriched in the flagellum of elongated spermatids, coinciding\nwith the period of intense cytoskeletal remodeling necessary for flagellar\nmorphogenesis. Moreover, our observation of partial co-localization between NSUN7\nand acetylated tubulin further supports a potential association of NSUN7 and the\ncentrosome. As mentioned above, we conducted experiments to identify the potential\ninteracting proteins of NSUN7 (Response Figure 4). It seems that NSUN7 may interact\nwith tubulin proteins, suggesting that NSUN7 may be associated with the centrosome.\nHowever, due to the high background in the detection method, based on our current\nresults, we cannot draw a conclusion that NSUN7 interacts with tubulin, nor identify\nwhich specific tubulin isoforms it may interact with. Additionally, it is difficult to\nspecifically determine whether the interaction between NSUN7 and tubulin requires the\npresence of RNA. Thus, the relationship between NSUN7 and the centrosome still\nrequires further investigation with high-sensitivity and high-resolution single molecular\napproaches.\nRegarding the reviewer’s valuable point about differentially expressed genes (DEGs)\nin spermatocytes, round spermatids, and elongated spermatids, we appreciate the\nsuggestion to consider their potential relevance despite their lower abundance\ncompared to elongated spermatids. We reanalyzed the single-cell RNA sequencing data\nand found that among the cilium organization-associated genes in elongated spermatids,\nTuba3a and Tuba3b showed the most significant changes (Now Figure 7D; Table S6).\nConsistently, RT-qPCR analysis using isolated elongated spermatids confirmed that the\nexpression levels of these two genes were markedly decreased (Now Figure 7F).\nNotably, these two tubulin isoforms are testis-specific, playing crucial roles in\nspermatogenesis and the maintenance of sperm morphology (Blake J, et al., Nucleic\nAcids Research, 2006; Akter M, et al., Scientific Reports, 2021). Together, there\nremains a possibility that NSUN7 is associated with centrosomal functions, which\nrequires further validation with advanced detection methods.\nAdditionally, our single-cell RNA-seq data show that Nsun7 mRNA is already\ndetectable in spermatocytes and round spermatids. Although we detected high NSUN7\nprotein level in elongated spermatids, due to the limited sensitivity of antibody-based\n10 / 18\ndetection, we cannot exclude the possibility that a small amount of NSUN7 protein also\nexists in spermatocytes and round spermatids. We hypothesize that NSUN7 may exert\npreparatory or regulatory functions that precede its structural role during spermatid\nelongation. These possibilities warrant further investigation using high-sensitivity and\nhigh-resolution detection technologies.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a compelling and well-structured study that addresses a long-\nstanding question regarding the function of NSUN7, a poorly characterized member of\nthe NSUN family of RNA methyltransferases. The authors provide strong evidence that\nNSUN7 lacks canonical catalytic activity due to key mutations in its SAM-binding\ndomain, distinguishing it from other NSUN proteins. They convincingly show that\nNSUN7 is enriched in elongated spermatids, and that its loss leads to defects in sperm\nmotility and axonemal structure, accompanied by downregulation of cilium-related\ntranscripts. These findings are significant and novel, as they establish NSUN7 as a non-\ncatalytic regulator with a potential structural or scaffolding role during spermatogenesis.\nHowever, the underlying molecular mechanism remains incompletely explored,\nparticularly in terms of RNA and protein interactions. Addressing the key questions\nbelow will be essential to fully define NSUN7’s biological role and support its proposed\nfunction in male fertility. To substantiate the novel model proposed—where NSUN7\nfunctions in a non-enzymatic, regulatory capacity—the manuscript would benefit\ngreatly from mechanistic data that clarify NSUN7’s molecular interactions and\nsubcellular dynamics. The following experiments and clarifications will strengthen the\nbiological conclusions and are likely essential for publication in a high-impact venue.\nResponse: We sincerely thank the reviewer for your insightful and constructive\ncomments. We fully agree that elucidating NSUN7’s molecular interactions and\nsubcellular dynamics is critical to substantiating its proposed non-enzymatic, regulatory\nrole. We have conducted experiments and revised the manuscript, Figures,\nSupplementary Figures and Tables, and we hope that these revisions could address the\nquestions you raised.\nMajor Comments:\n1. Is NSUN7's localization RNA-dependent in human and mouse samples? The authors\nshould assess whether RNase treatment alters NSUN7 localization, using\nimmunostaining or fractionation. This would help determine if RNA binding anchors\nNSUN7 to specific compartments. And whether NSUN7 localization is conserved in\nmouse and human.\n11 / 18\nResponse: We sincerely thank the reviewer for this insightful and valuable comment.\nTo address this point, we performed nuclear-cytoplasmic fractionation of adult mouse\ntestes. The results showed that NSUN7 is mainly localized in the cytoplasmic fraction,\nwith Lamin A/C and GAPDH serving as nuclear and cytoplasmic markers, respectively\n(Response Figure 2). RNase A treatment did not markedly alter the distribution of\nNSUN7 between nuclear and cytoplasmic fractions, suggesting that RNA digestion\ndoes not significantly affect its overall subcellular localization at this level of resolution.\nTo achieve a more precise mapping of NSUN7’s subcellular localization, methods such\nas immunoelectron microscopy or advanced super-resolution imaging would be more\nrefined approaches. However, despite extensive optimization and repeated attempts,\nour currently available antibody did not provide sufficient sensitivity and specificity for\nhigh-resolution subcellular visualization.\nResponse Figure 2\nNevertheless, encouraged by the reviewer’s suggestion, we next explored how\nRNase treatment could affect the NSUN7’s property. Specifically, we treated mouse\ntestis lysates with RNase A before immunoblotting, and found that the endogenous\nNSUN7 protein level decreased markedly after RNase A digestion (Now Figure S9A,\nas shown below). A similar phenomenon was observed when human NSUN7 was\noverexpressed in cultured cells: RNase A treatment resulted in a reduction in the\ndetectable NSUN7 protein level (Now Figure S9B, as shown below). Our results\nsuggest that the association of NSUN7 with RNA contributes to maintaining its protein\nstability. Although these results do not yet provide direct evidence for RNA-dependent\nlocalization at the microscopic level, they nonetheless point to an intriguing possibility\nof an association between NSUN7 and RNA. We have now included this new set of\ndata and the corresponding interpretation in the revised manuscript (Page 19, Lines 27-\n33).\n12 / 18\n2. Is NSUN7 expression paterns conserved in human tissues?\nResponse: Thanks for your meaningful and insightful suggestion. According to the\nHuman Protein Atlas database (Uhlén M, et al., Science, 2015; Karlsson M, et al.,\nScience Advances, 2021), human NSUN7 is predominantly enriched in the testis among\n55 human tissues (Response Figure 3A). We examined the underlying data and found\nthat these testis samples are primarily derived from adult males. This observation is\nconsistent with our findings that mouse Nsun7 is mainly enriched in the adult mouse\ntestis.\nAt the single-cell level, human NSUN7 is broadly expressed in spermatocytes, early\nspermatids, and late spermatids, with the highest expression observed in late spermatids\n(Response Figure 3B), which is consistent with the expression pattern of mouse Nsun7\nrevealed by our single-cell RNA sequencing. Furthermore, human NSUN7 is also\nexpressed in ciliated cells across multiple tissues, including the lung, fallopian tube,\nendometrium, and bronchus (Response Figure 3B). Consistently, our analysis of\nsingle-cell RNA sequencing data from mouse ovary and fallopian tube (Qin G, et al.,\niScience, 2022; Response Figure 1) reveals that mouse Nsun7 is also expressed in the\nepithelial cells of the fallopian tube. Collectively, these observations show that the\nexpression pattern of NSUN7 is conserved between human and mouse.\n13 / 18\nResponse Figure 3\n(A) Expression level of NSUN7 in different human tissues.\n(B) Expression level of NSUN7 in different human single cell types.\n3. Does NSUN7 retain RNA-binding ability despite lacking methyltransferase activity?\nRIP or iCLIP using epitope-tagged NSUN7 in spermatids or other cell types would\nclarify its RNA targets and support a scaffolding or post-transcriptional regulatory role.\nResponse: We sincerely thank the reviewer for raising this important mechanistic\nquestion. We have made extensive efforts to experimentally address this issue.\nSpecifically, we tested antibodies generated from more than ten self-designed peptide\nantigens as well as all commercially available antibodies, but none successfully\ndetected endogenous NSUN7 in testis lysates. Our in-house-generated antibody, raised\nagainst the N-terminal domain of NSUN7, can be used for immunofluorescence and\nWestern blotting; however, despite multiple attempts, it has not yet yielded successful\nresults in immunoprecipitation, RIP, or iCLIP experiments. However, despite repeated\noptimization, this antibody has not yet yielded positive signals in these assays, making\n14 / 18\nit technically challenging to capture RNAs bound by endogenous NSUN7.\nDuring the preparation of this manuscript, Guseva E, et al. generated HA-tagged\nNSUN7 mice, and using anti-HA antibody to perform CLIP in mouse testis and found\nthat NSUN7 binds specific mRNAs, with one-third of these mRNAs being specific to\nelongated spermatids, supporting its potential RNA-binding capability. Our results\nshow that RNase treatment had an impact on NSUN7 protein stability (Now Figures\nS9A, B), further indicating that the association of NSUN7 with RNA contributes to\nmaintaining its protein stability. Nevertheless, it remains uncertain whether the reduced\nexpression of cilium organization-associated genes results from this potential RNA-\nrelated role. Accordingly, we have revised the relevant part of the Discussion (Page 22,\nLines 18-30).\n4. Determining NSUN7’s protein interactors, especially in relation to microtubule\nproteins at the flagella, is critical. Co-immunoprecipitation followed by mass\nspectrometry using tagged NSUN7 could uncover partners relevant to its structural role\nin sperm motility.\nResponse: Thanks for your valuable suggestion. We conducted co-\nimmunoprecipitation followed by mass spectrometry to detect NSUN7-interacting\nproteins under treatment with or without RNase A. Flag-tagged mouse NSUN7 was\noverexpressed in cells. Then, the cells were combined with mouse testis and lysed\ntogether, and Flag-IP MS assays were used to pull down potential NSUN7-interacting\npartners (Response Figure 4A). The MS results showed a significant increase in β-\ntubulin and α-tubulin proteins in the Flag-tagged NSUN7 pull-down samples compared\nto the control group (Response Figures 4B-D). However, after RNase A treatment, the\ntubulin signal intensity in NSUN7 IP samples became comparable to that in the control\nIP samples (Response Figures 4B-D), indicating that NSUN7 may interact with tubulin\nproteins in the presence of RNA.\nHowever, when validating the IP samples, we observed high tubulin binding\nbackground in both control and NSUN7 IP groups. Moreover, RNase A treatment led\nto an increase in the non-specific binding of tubulin (Response Figure 4E). After\nRNase A treatment, NSUN7 seems not to bind tubulin, but other possibilities may exist:\ni) The reduced NSUN7 protein levels by RNase A treatment may weaken its interaction\nwith tubulin; ii) increased non-specific binding of tubulin may mask the interaction\nsingal. Together, we cannot conclude that NSUN7 interacts with tubulin, nor identify\nwhich specific tubulin isoforms it may interact with. The potential interacting partners\nof NSUN7, the role of RNA in this process, and NSUN7-involved signaling pathways\nstill await further validation with high-sensitivity and high-resolution approaches.\n15 / 18\nResponse Figure 4\n(A) IP assays of NSUN7-Flag. The empty vector was transfected as a negative control.\n(B) MS detection of potential interacting proteins of NSUN7. Color was applied to features\nwith a log2fold change of intensity ratios greater than 1 between groups.\n(C) and (D) Under treatment with or without RNase A, NSUN7 and tubulin proteins detection\nintensity in the IP samples was analyzed by mass spectrometry.\n(E) Detection of α-tubulin signal in the IP samples.\n5. Since human infertility has been linked to point mutations in NSUN7, the authors\nshould discuss or experimentally test whether such mutations disrupt NSUN7’s\ninteraction with key structural proteins, contributing to defective sperm motility.\nResponse: Thanks for your valuable recommendation. Khosronezhad N, et al.\n(Reproduction, Fertility and Development, 2015a) identified two mutations in exon 7\nof the NSUN7 gene—the C26232T transition and T26248G transversion—that are\nassociated with reduced sperm motility in Iranian men with asthenospermia. The\nC26232T transition is a silent mutation, changing codon GGC to GGU, both of which\n16 / 18\nencode Gly. In contrast, the T26248G transversion is a missense mutation that leads to\nthe substitution of Ser (encoded by UCC) with Ala (encoded by GCC). Additionally,\nKhosronezhad N, et al. (J. Assist. Reprod. Genet., 2025b) also reported an A11337-\ndeletion mutation in exon 4 of asthenospermic men, which causes a frameshift and\npremature termination of the NSUN7 protein, resulting in a truncated protein of only\n156 amino acids.\nWe constructed plasmids containing the relevant human infertility-related NSUN7\npoint mutations and examined the expression of these mutants. Our results showed that\ncompared to the wild-type, the C26232T transition and T26248G transversion mutants\nled to reduced protein levels of NSUN7, while no protein production was detected for\nthe A11337-deletion mutation. Thus, we concluded that although the C26232T\ntransition and T26248G transversion cause a single amino acid substitution of Ser to\nAla in position 308, they may reduce NSUN7 protein levels. The A11337-deletion\nmutation completely abolishes NSUN7 protein expression. Therefore, these point\nmutations lead to varying degrees of loss in NSUN7 protein levels. (Now Figure S9C,\nas shown below). We also described this result on current Page 19, Lines 34-39 and\nPage 20, Lines 1-4.\nMinor Comments:\nFigure 2B: Labels for 18S/WT and 18S/KO are missing in the bar chart and should be\nadded for clarity.\nResponse: Thank you very much for pointing out this mistake. We have corrected the\nx axis labeling in Figure 2B.\n17 / 18\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We are grateful for the reviewer's valuable and insightful suggestions. We\nhave conducted experiments and revised the manuscript, Figures, Supplementary\nFigures and Tables. We hope that our revised manuscript now offers a more\ncomprehensive elucidation of the molecular mechanism of NSUN7.\n18 / 18"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my issues and those of the other reviewers, and have improved the manuscript. I do not have additional comments.", "reviewer_2": "The authors have thoroughly and effectively addressed the comments and questions from both this reviewer and the co-reviewer. We have no further questions. This is an excellent manuscript. Congratulations to the authors!", "reviewer_3": "The authors have addressed all our comments by providing new experimental data, additional analyses, and revised interpretations. They investigated NSUN7’s RNA-dependent localization, confirming that its cytoplasmic distribution is largely unchanged after RNase treatment but that RNA association contributes to protein stability. They demonstrated that NSUN7 expression patterns are conserved between mouse and human, explored its potential RNA-binding ability despite technical limitations, examined possible protein interactors such as tubulins through co-immunoprecipitation and mass spectrometry, and analyzed human infertility-related NSUN7 mutations showing reduced or abolished protein expression.\nOverall, the authors have comprehensively responded to all reviewer concerns.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter