Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster

Kaneko, Shunya; Miyoshi, Keita; Tomuro, Kotaro; Terauchi, Makoto; Tanaka, Ryoya; Kondo, Shu; Tani, Naoki; Ishiguro, Kei-Ichiro; Toyoda, Atsushi; Kamikouchi, Azusa; Noguchi, Hideki; Iwasaki, Shintaro; Saito, Kuniaki

doi:10.1038/s41467-024-52389-0

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Published: 24 September 2024

Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster

Nature Communications volume 15, Article number: 8147 (2024) Cite this article

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Abstract

Modification of guanosine to N⁷-methylguanosine (m⁷G) in the variable loop region of tRNA is catalyzed by the METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) causes no major effect on the development of non-gonadal tissues, but abolishes the production of elongated spermatids and mature sperm, which is fully rescued by expression of a Mettl1-transgene, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m⁷G is required for spermatogenesis. Mettl1-KO results in a loss of m⁷G modification on a subset of tRNAs and decreased tRNA abundance. Ribosome profiling shows that Mettl1-KO led to ribosomes stalling at codons decoded by tRNAs that were reduced in abundance. Mettl1-KO also significantly reduces the translation efficiency of genes involved in elongated spermatid formation and sperm stability. Germ cell-specific expression of Mettl1 rescues disrupted m⁷G tRNA modification and tRNA abundance in Mettl1-KO testes but not in non-gonadal tissues. Ribosome stalling is much less detectable in non-gonadal tissues than in Mettl1-KO testes. These findings reveal a developmental role for m⁷G tRNA modification and indicate that m⁷G modification-dependent tRNA abundance differs among tissues.

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Introduction

Post-transcriptional epigenetic modifications of RNA are together known as the “epitranscriptome” and are being revealed to have diverse and widespread functions in gene regulation. To date, over 170 RNA modifications have been identified, approximately 80% of which are found in tRNAs^1,2. Chemical modifications regulate tRNA structure, dynamics, and interactions with other molecules, and result in altered translation^3,4. While some tRNA modifications play a fundamental role in cell proliferation, an increasing number of studies show that aberrant tRNA modification can result in severe developmental defects and human diseases, indicating that tRNA modifications play roles in specific tissues during animal development^4,5,6,7.

N7-methylguanosine (m⁷G) is an RNA modification observed in mRNA caps⁸ and at internal positions within tRNAs, rRNAs, mRNAs and miRNAs^{9,10,11,12,13,14,15}. m⁷G in tRNAs is widely conserved in prokaryotes, eukaryotes and some archaea¹⁶. In eukaryotes, Methyltransferase-like 1 (METTL1) catalyzes the addition of m⁷G at internal sites of tRNAs, mRNAs, and miRNAs^10,12,15,17. METTL1 interacts with the non-catalytic subunit WD repeat domain 4 (WDR4)^{17,18,19,20,21}, and both are widely conserved from yeast to mammals. m⁷G modification occurs at the 46th guanosine in the variable loop region of tRNAs, within the conserved motif, “RAGGU” (R = A or G, G represents m⁷G)¹⁰. In Saccharomyces cerevisiae, transfer RNA methyltransferase 8 (Trm8), a homolog of METTL1, introduces the m⁷G modification onto tRNAs, such as tRNA ValAAC, and promotes tRNA stability and cell growth at higher temperatures^17,18,22. Hypomodified tRNAs are degraded by the 5′–3′ exonucleases, Rat1 and Xrn1, which form the rapid tRNA decay (RTD) pathway in yeast^22,23,24. Rat1 and Xrn1 are highly conserved among animals, indicating that a similar degradation system is present in animals²⁵. Indeed, METTL1-depletion in mammalian cells leads to decreased steady-state levels of several tRNAs that are targets of METTL1, retardation of cell growth^26,27 and impaired translation of genes associated with neural differentiation¹⁰. Conversely, increased levels of METTL1 result in increased m⁷G modification of specific tRNAs, such as tRNA ArgTCT, tRNA stabilization, cell cycle acceleration through enhanced translation of cell cycle related genes, such as CDK6 and CDK8, and promotion of oncogenicity²⁷. The primary let-7e miRNA is also methylated by METTL1¹². Internal m⁷G modifications in mRNAs are recognized by Quaking proteins, which enable their transport into stress granules. This modulates mRNA stability and the cellular stress response in mammals²⁸. These studies have revealed the role of m⁷G at the single cell level; however, its roles at tissue and organ levels during animal development are less well characterized.

Recent clinical studies have revealed that variations in METTL1 and WDR4 cause microcephalic primordial dwarfism, Galloway–Mowat syndrome^29,30,31, multiple sclerosis^32,33, and male infertility³⁴, indicating a potential link between internal m⁷G regulation and pathological characteristics³⁵. Furthermore, knockout of Wdr4 (a mouse homolog of Drosophila melanogaster Wuho (Wh) in mice results in embryonic lethality³⁶. These observations indicate that the m⁷G modification machinery is critical for mammalian development; however, it is unclear whether m⁷G modification alone is responsible for the above developmental defects. Indeed, WDR4 physically interacts with proteins other than METTL1. WDR4 is proposed to be involved in genome stability through interaction with Flap endonuclease 1³⁶ and in cerebellar development and locomotion through interaction with ATRGAP17³⁷. Therefore, it is not known whether the developmental disorders caused by METTL1 or WDR4 variants are directly linked to defective m⁷G modification.

In D. melanogaster, Wh is required for gonad development^38,39. Wh mutants show sterility with arrested spermatogenesis in males and semi-sterile phenotypes with abnormal egg chamber and decreased germline stem cell number in females^38,39. Wh is considered to control ovary function through interaction with partner proteins, including Mei-P26, Nanos, and Bgcn³⁸. However, it remains to be addressed whether Wh associates with the Drosophila METTL1 ortholog and if the fertility defects in Wh mutants are caused by a loss of m⁷G modification or via defective association with interactors of Wh other than Mettl1.

Here, we demonstrated that candidate gene 4045 (CG4045) (hereafter referred to as Mettl1) is required for Drosophila male and female fertility. Mettl1 associates with Wh in vivo and in vitro and catalyzes m⁷G-modification on a subset of tRNAs in a Wh-dependent mechanism. Depletion of Mettl1 results in a loss of m⁷G modification and decreased abundance of nearly all of the tRNAs targeted by Mettl1. Consistent with the decrease in tRNA abundance, we found increased occurrence of ribosome pausing and subsequent collisions of trailing ribosomes in Mettl1-KO testes at the codons decoded by the reduced tRNAs, leading to the reduction of the translation efficiency of genes involved in mature sperm formation. Taken together, our work reveals the developmental role of Mettl1-dependent m⁷G in multicellular organisms.

Results

Mettl1, the Drosophila ortholog of the m⁷G-modifier, METTL1, is essential for Drosophila fertility

We studied the physiological functions of Mettl1 in D. melanogaster. The Drosophila gene, CG4045 (herein referred to as Mettl1), is an ortholog of mammalian METTL1 and contains an RNA methyltransferase-like domain that is highly conserved across animals (Fig. 1a, Supplementary Fig. 1a)⁴⁰. To generate a Mettl1-knock out (KO) strain using the CRISPR/Cas9 system, a guide RNA (gRNA) was selected to target the N-terminal part of Mettl1 (Fig. 1a). Two different transgenic strains harboring either a 1 nucleotide deletion (Mettl1^KO1) or a 23-nucleotide deletion with an 8-nucleotide insertion (Mettl1^KO2) were generated, both of which lose a large part of the methyltransferase-like domain because of frame-shift (Fig. 1a, Supplementary Fig. 1b). To detect Mettl1 and the Mettl1-interacting partner, Wh, mouse-monoclonal antibodies were generated. Western blot analyses showed a strong ~32 kDa Mettl1 signal in heterozygous ovaries and control (yw) testes (Fig. 1b, c). In contrast, this signal was undetectable in Mettl1^KO1 and Mettl1^KO2 ovaries and Mettl1^KO1 testes (Fig. 1b, c, Supplementary Fig 1c), indicating that Mettl1 was absent in Mettl1-KO animals. Notably, a Wh signal was still observed in Mettl1-KO flies (Fig. 1b), indicating that Wh stability is unaffected by the absence of its heterodimer partner, Mettl1.

**Fig. 1: Mettl1 is essential for maintaining fertility.**

Basic analyses of adult morphology (Supplementary Fig. 1d), body weight (Supplementary Fig. 1e), rate of emerging adult flies (Supplementary Fig. 1f) and locomotive activity (Supplementary Fig. 1g), indicated that Mettl1^KO1 and Mettl1^KO2 flies developed normally to the adult stage. However, we observed that mutant males and females had significantly reduced fertility (Fig. 1d, e, Supplementary Fig. 2a–c). Mutant males produced one tenth and mutant females one third of the progeny produced by control flies (Fig. 1d, e, Supplementary Fig. 2a–c). Consistent with the reduced fertility of Mettl1-KO females, the size of Mettl1-KO ovaries was significantly reduced (Supplementary Fig. 2d). To confirm whether this was caused by loss of Mettl1 function, we introduced the Mettl1 genomic region into the second chromosome. The [Mettl1] transgene rescued fertility and ovary size to the levels of the control groups (Fig. 1d, e, Supplementary Fig. 2b, d). Moreover, to evaluate the maternal contribution to fertility, we generated maternal and zygotic mutant males. Small numbers of offspring (less than 10) were detected in three of seven crosses from zygotic mutant males, whereas no offspring were produced by maternal and zygotic mutant males, indicating a maternal contribution of Mettl1 (Fig. 1f). Taken together, these data reveal a critical role of Mettl1 in male fertility.

Mettl1 depletion impairs the formation of elongated spermatids and results in the loss of mature sperm

Drosophila spermatogenesis involves germ cells communicating with somatic cells, proceeding through mitotic and meiotic cell division, differentiating into elongating spermatids, and forming bundle structures in the testis. Each step in this process is tightly regulated (Fig. 2a)⁴¹. To study the expression patterns of Mettl1 in the testis, we generated a transgenic strain carrying C-terminally Flag-tagged Mettl1, [Mettl1-Flag] (Fig. 2b) and confirmed that [Mettl1-Flag] rescued Mettl1^KO1 fertility to a similar extent as untagged [Mettl1] (Fig. 1d, Supplementary Fig. 3a). We observed Flag-tag signals in almost all testicular cells, indicating that Mettl1 is ubiquitously expressed in the various gonadal cell types (Fig. 2b). Wh-GFP shows a similar ubiquitous expression pattern through spermatogenesis, indicating the presence of the Mettl1/Wh heterodimer in all testicular cells³⁹. To study whether spermatogenesis was affected by Mettl1-KO, we assessed mature sperm generation. We found no mature sperm in the seminal vesicles of Mettl1-KO testes (Fig. 2c), which is consistent with the decreased fertility of Mettl1-KO males (Fig. 1d). To examine the spermatogenesis defects in more detail, we studied the germ cell marker proteins, Aubergine, a marker of germline cells, and Boule, a Drosophila orthologue of vertebrate DAZL, a marker of 32- and 64-cell cysts and elongating spermatids within Drosophila testes⁴². Aubergine was observed in a wild-type pattern within Mettl1-KO testes (Supplementary Fig. 3b). Moreover, Boule was also detected in a wild-type pattern in 32- and 64-cell cysts within Mettl1-KO testes (Fig. 2d, Supplementary Fig. 3c). Boule-positive cysts defective in elongation were accumulated near the seminal vesicle region (Fig. 2d), indicating that the loss of Mettl1 did not affect meiosis but may have affected the elongation step of spermatogenesis. Furthermore, we observed no elongated spermatids (Fig. 2e). We then examined the distribution of acetylated microtubules, a marker of elongated spermatids⁴³. Most Mettl1-KO testes (23/31) showed a decreased acetylated microtubule signal (indicating an elongation defect, Fig. 2f). The remainder had no elongated spermatids (8/31, Fig. 2f). Together, these results indicate that elongated spermatid formation and the later stages of spermatogenesis are severely impaired in Mettl1-KO testes.

**Fig. 2: Loss of Mettl1 results in absence of mature sperm in the testis.**

We observed no change in overall morphology or in the distribution of somatic niche cells (hub cells) or somatic cyst cells in Mettl1-KO testes, indicating the requirement for Mettl1 in testicular germ cells (Supplementary Fig. 3d). Mettl1 is still detected in the carcasses of males after gonad removal (Supplementary Fig. 3e), indicating that Mettl1 may play a critical role in elongated spermatid formation in tissues other than the testis. We assessed the copulation behavior of Mettl1-KO males and tumorous testis (tut)-KO males, which are also deficient in spermatogenesis⁴⁴. Interestingly, both Mettl1-KO and tut-KO males showed significantly decreased copulation rate (Supplementary Fig. 3f), indicating that disrupted spermatogenesis impairs sexual behaviors via unknown signaling from the testes to the brain. To directly examine whether Mettl1 acts in a cell-autonomous or non-cell autonomous manner, we prepared a Mettl1-transgene driven by a germ-specific Nanos promoter and conducted a fertility assay. The Nanos-Mettl1-transgene [nosP-Mettl1] fully rescued the sterile phenotype and acetylated microtubule signal of Mettl1-KO males (Fig. 2g, Supplementary Fig. 3g). Moreover, somatic cell-specific knockdown (KD) of Mettl1 did not affect fertility (data not shown). Taking these findings together, we concluded that Mettl1 plays a critical role in elongating spermatid formation via a cell-autonomous mechanism.

Catalytic activity of Mettl1 is required for Drosophila fertility

Although mammalian METTL1 and its yeast ortholog, Trm8, catalyze the m⁷G modification on tRNAs, it is not known whether Drosophila Mettl1 possesses this activity^10,17. Given that METTL1 acts as the catalytic partner in a heterodimer with WDR4^10,17,20,21, we investigated the formation of a homologous complex in flies. We conducted immunoprecipitation from [Mettl1-Flag] ovaries or Oregon R wild-type ovaries using two different buffer conditions and performed liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the samples (Fig. 3a, Supplementary Fig. 4a, b). Mettl1-Flag co-immunoprecipitated with Wh irrespective of buffer conditions, indicating that the Mettl1/Wh heterodimer is formed in vivo, as in other organisms (Fig. 3a, Supplementary Fig. 4a, b). Notably, we did not detect Mei-P26, Nanos or Bgcn proteins, which can associate with Wh³⁸, indicating that Wh makes a distinct protein complex with either Mettl1 or these other proteins. To confirm direct interaction, we purified MBP-Mettl1 and GST-Wh from Escherichia coli and performed in vitro GST-pull down assays (Supplementary Fig. 5a, b). MBP-Mettl1 but not MBP alone associated with GST-Wh but not with the GST moiety (Supplementary Fig. 5a, b).

**Fig. 3: Catalytic activity of *Drosophila* Mettl1 is required for fertility.**

We then conducted in vitro m⁷G methylation assays using the above recombinant proteins with a synthesized tRNA and a shortened tRNA fragment designed according to a previous study⁴⁵, both of which include the RAGGU motif, a conserved target sequence of eukaryotic METTL1 (Fig. 3b, Supplementary Fig. 5c, d). Drosophila Mettl1 required Wh for tRNA methylation, consistent with a recently proposed structural model of m⁷G methylation (Supplementary Fig. 5d)^20,21. Mettl1 did not introduce m⁷G to a target RNA in which the 4th guanosine of the RAGGU motif was converted to cytosine, indicating that Drosophila Mettl1/Wh methylates target tRNAs in a similar manner to that of METTL1/WDR4 in other eukaryotes (Supplementary Fig. 5d)¹⁰. To create a catalytic dead mutant of Mettl1, L157A and D160A mutations (Mettl1-Cd: Cd), both of which are located in the methylation consensus motif, were engineered (Supplementary Fig. 1a). We confirmed that these mutations abolished methylation activity of Mettl1 in vitro (Fig. 3b).

This finding led us to test the requirement of Mettl1 catalytic activity for fertility. We prepared a catalytic dead Mettl1-transgene [Mettl1-Cd], harboring L157A and D160A mutations in Mettl1 (Fig. 3c) and conducted fertility rescue experiments (Fig. 3c). The sterile phenotype of Mettl1^KO1 was rescued by the wild-type Mettl1-transgene [Mettl1], but not by [Mettl1-Cd], indicating that male fertility is dependent on the catalytic activity of Mettl1 (Fig. 3c). [Mettl1-Cd] transgene shows slightly lower levels of the mutant protein than wild-type (Supplementary Fig. 5e). Thus, we can not rule out the possibility that the failure of the rescue by [Mettl1-Cd] transgene is due to a slight level of difference of the mutant proteins. [Mettl1-Cd] did not show any dominant-negative effects on fertility in wild-type backgrounds (Supplementary Fig. 5f).

Drosophila Mettl1 mediates m⁷G modification of tRNA

To understand the molecular mechanism underlying the Mettl1-KO fertility defects, we surveyed m⁷G modification sites on tRNAs because tRNAs are efficiently methylated by METTL1 in mammalian cells¹⁰. We performed an m⁷G site-specific cleavage assay using sodium borohydride (NaBH₄) and aniline^46,47. The resulting 5′ and 3′ fragments were detected by northern blotting using specific tRNA probes (Fig. 4a, b, Supplementary Fig. 6a, b). We detected 3′ fragments of tRNAs isolated from testes (Fig. 4b) and ovaries (Supplementary Fig. 6a) of wild-type Mettl1 but not Mettl1-KO flies, supporting Mettl1 deposition of m⁷G onto tRNAs in both tissues (Fig. 4b, Supplementary Fig. 6a). Moreover, the 3′ fragments from the same tRNAs were lost in Wh mutant testes (Fig. 4c), consistent with the collaborative role of Wh for m⁷G modification of tRNA in vitro (Fig. 4c, Supplementary Fig. 5c).

**Fig. 4: *Drosophila* Mettl1 methylates tRNA in gonads.**

We next comprehensively investigated the cleaved tRNA fragments by deep sequencing [i.e., tRNA reduction and cleavage sequencing (TRAC-seq)^10,48] (Fig. 4d, Supplementary Fig. 6c). TRAC-seq identified 23 tRNAs in testes and 22 tRNAs in ovaries. In our analysis, tRNA ArgTCT was categorized as m⁷G-modified in testis but not in ovary. This difference can be attributed to the limited number of tRNA reads from the Drosophila ovary. The m⁷G-modified tRNAs have the RAGGU motif (Fig. 4f) located in the variable loop (Fig. 4g), which is similar to the findings of a previous study¹⁰. Consistent with the northern blot data (Fig. 4b, c), the TRAC-seq cleavage score was markedly decreased in Mettl1-KO samples, further supporting the function of Mettl1 in m⁷G modification of tRNA (Fig. 4h). Together, these data indicate that Mettl1/Wh is responsible for introducing the m⁷G modification onto a subset of tRNAs in the testis.

Drosophila Mettl1 regulates the abundance of a subset of tRNAs

In S. cerevisiae, loss of m⁷G modification on tRNAs results in decreased tRNA abundance^17,22. This mechanism for this is the rapid tRNA decay (RTD) pathway mediated by exonucleases^22,23,24. We therefore reasoned that a similar RTD pathway acts in Drosophila upon the loss of Mettl1. In testes, Mettl1 depletion resulted in changes in the levels of some tRNAs (Fig. 5a, b). In Drosophila testis, the mean steady-state levels of 15 m⁷G-modified tRNAs (ProAGG, ProCGG, ProTGG, ValAAC, ValCAC, ValTAC, LysCTT, LysTTT, ArgTCT, AlaAGC, AlaCGC, AlaTGC, CysGCA, TyrGTA, and iMetCAT) were significantly decreased, whereas the abundances of non-m⁷G modified tRNAs (22 of 45 tRNAs) showed limited changes (Fig. 5a). We divided m⁷G-modified tRNAs into two groups according to steady-state levels of tRNA abundance in Mettl1-KO testes (Fig. 5b, c, Supplementary Fig. 7a): destabilized in Mettl1-KO (group 1) and unaltered in Mettl1-KO (group 2). We also defined m⁷G-hypomodified tRNAs as group 3 (Fig. 5a, c, Supplementary Fig. 7a). Consistent with the TRAC-seq data (Fig. 5a–c), northern blot analysis validated the reduced levels of group 1 tRNAs (ProTGG and ValCAC) and the absence of change in group 2 tRNA (TrpCCA) and group 3 tRNA (LeuAAG) in Mettl1-KO testes (Fig. 5d) (see Fig. 6 for further characterization of these tRNA groups for translation).

**Fig. 5: *Drosophila* Mettl1 is required to maintain tRNA abundance in the testis.**

**Fig. 6: Ribosome profiling in *Mettl1*^KO1 testes.**

To gain insight into how the levels of hypomodified tRNAs are decreased in testes, we examined aminoacylation levels of tRNAs by acid urea polyacrylamide gel electrophoresis and northern blot analysis. We found that the majority of tRNA ValCAC, an m⁷G-tRNA, was aminoacylated in Mettl1-KO testes, indicating that the decrease of tRNA in Mettl1-KO testes is not caused by loss of aminoacylation (Supplementary Fig. 7b). The reduced levels of a subset of tRNAs in Mettl1-KO flies also led us to investigate whether the RTD pathway explains the sterile phenotype. A genetic approach revealed that Trm8, a METTL1 ortholog in S. cerevisiae, is important for m⁷G tRNA modification and stability and for temperature sensitive growth^18,22. It is noteworthy that the RTD pathway can be suppressed by overexpression of eukaryotic translational elongation factor 1 A (eEF1A), encoded by the TEF1 gene in yeast, because eEF1A binds directly to tRNAs and protects them from the RNA decay machinery^49,50. Therefore, we asked whether overexpression of eukaryotic translational elongation factor 1 alpha 1 (eEF1α1, CG8280), a fly homolog of eEF1A, rescues the male sterility in Mettl1-KO flies (Supplementary Fig. 7c). Actin mRNAs are highly expressed in various cell types including in testes; therefore, we expressed eEF1A under the control of Act-Gal4. Overexpression of eEF1α1 partially rescued the fertility of Mettl1-KO males, indicating that a pathway similar to the S. cerevisiae RTD pathway may play a role and regulate tRNA abundance in Drosophila.

In mammalian A549 cells, let-7e-5p miRNA biogenesis is affected by m⁷G methylation¹² because m⁷G modification in the primary transcript of let-7e disrupts G-quadruplex formation and facilitates precursor miRNA processing¹². In D. melanogaster, depletion of let-7 causes a spermatogenesis defect⁵¹; therefore, the phenotype that we observed in Mettl1-KO flies can be theoretically explained by a defect in let-7 biogenesis. However, the steady-state level of let-7 miRNA was not significantly changed in Mettl1-KO testes, indicating that the sterile phenotype of Mettl1-KO males is not caused by m⁷G modification of the let-7 primary miRNA (Supplementary Fig. 7d). Notably, sequence comparison between mouse and D. melanogaster indicated that the RAGGU motif in the mouse let-7e primary miRNA is not identical to that in D. melanogaster (UAGGU) and that the G bases that make up the G-quadruplex are A bases in D. melanogaster. This might explain why let-7 was not m⁷G-modified in Drosophila (Supplementary Fig. 7e). Taking these observations together, we conclude that the Drosophila let-7 primary miRNA is not modified by Mettl1 and is unrelated to the sterile phenotype of Mettl1-KO males (Fig. 2, Supplementary Fig. 7d).

Translatome analysis in Mettl1-KO testis

To examine whether the reduced abundance of tRNAs affects mRNA translation, we performed ribosome profiling of dissected testes^52,53. Given that dissected testes provide limited material, we employed a method tailored for low input with RNA-dependent RNA amplification⁵⁴. Remarkable 3-nt periodicity (Fig. 6a) and high reproducibility (Fig. 6b, c) confirmed high quality data were obtained from the small amounts of tissue. We observed remarkably high ribosome occupancy on A-site codons with the loss of Mettl1 (Fig. 6d). Many of these codons corresponded to codons decoded by the m⁷G-modified tRNAs (Fig. 6d). Notably, among the m⁷G-modified tRNAs, the sub-group with reduced abundance (group 1, Fig. 5c) showed prominent effects on ribosome traversal (Fig. 6e). In support of these findings, ribosome profiling of testes from another Mettl1-KO (Mettl1^KO2) showed similar results (Supplementary Fig. 8a–d). Strikingly, ribosome pausing on codon CCA, recognized by group 1 tRNA ProTGG, induced collisions with trailing ribosomes to form disomes and trisomes (Fig. 6f). We observed that more pronounced ribosome stalling on CCA, GUG, CCU, and CCG codons occurred on a subset of transcripts, such as ones encoding genes essential for fertility (Fig. 6g, Supplementary Table 1).

In addition to the codon-wise assessment, we also investigated the impact of m⁷G tRNA modification on translation efficiency across open reading frames (ORFs). We measured translation efficiency (TE) after normalizing the ribosome footprints against RNA abundance measured by RNA-Seq and found that TE for a notable number of transcripts (1078) was down-regulated by the loss of METTL1 (Fig. 7a). Although these TE down-regulated transcripts were more prone to possess codons decoded by group 1 tRNAs (Fig. 7b), we did not detect a correlation between genes that were downregulated in TE and their Pro codon usage (Supplementary Fig. 9a). Gene ontology (GO) terms associated with the TE down-regulated genes included the key terms for gonadal functions, such as sperm individualization, sperm axoneme assembly, and spermatid development (Fig. 7c), consistent with the observed phenotype of Mettl1-KO. We noticed translational impairment of Don Juan (Dj), which is expressed in elongated spermatids and individualized sperms⁵⁵, in Mettl1-KO testis (Fig. 7d). We validated this observation using a GFP-tagged Dj transgene; Dj-GFP expression was significantly decreased in Mettl1-KO testis (Fig. 7d) compared with a control GFP-tagged transgene (Vasa-GFP, Supplementary Fig. 9b).

**Fig. 7: Analysis of TE in testes and comparative analysis with non-gonadal tissues.**

In addition to transcripts with down-regulated TE, we found a small fraction of transcripts with higher TE in Mettl1-KO testes (Fig. 7a). These mRNAs included Crc, a Drosophila homolog of ATF4, which has an upstream ORF (uORF) (Supplementary Fig. 9c)⁵⁶. The uORF typically traps scanning ribosomes and inhibits the complex reaching the main downstream ORF; however, reduced abundance of the initiator tRNA (Fig. 5b) may explain the induction of leaky scanning of the uORF, as represented by eIF2 inactivation in the integrated stress response⁵⁷. Taken together, these data indicate that translational changes in the late stage of spermatogenesis lead to infertility of Mettl1-KO males (Fig. 1d).

Phenotypic analyses showed that Mettl1-KO had no major effect on development except for in gonads (Supplementary Fig. 1d–g). We, therefore, investigated whether this phenotype correlates with tRNA modification status and TE in testes and non-testes tissue. We prepared body samples without testes (body) and analyzed the status of m⁷G-tRNA modification (Fig. 7e). As expected from the Mettl1 protein levels in body samples (Supplementary Fig. 3e), the 3′ shorter fragment was observed at similar extents in body and testis samples in a Mettl1-dependent manner (Fig. 7e). However, loss of Mettl1 had little effect on ribosome stalling in body samples. Ribosome occupancy change at CCA codons in body samples was remarkably lower than that found in testis (for example, as log2 ratio, 2.1 vs. 0.6 in Mettl1^KO1 and 2.4 vs. 0.6, in Mettl1^KO2) (Supplementary Fig. 9d–f), indicating that ribosome stalling is more prominent in testis compared with non-gonadal tissues. Notably, Nanos-driven Mettl1 failed to rescue the status of m⁷G modification in body samples (Fig. 7e) but recovered the 3′ shorter fragment and tRNA Pro abundance in testis (Fig. 7e, f), further supporting the critical role of Mettl1 in male fertility via a cell-autonomous mechanism (Fig. 2g).

Discussion

Here, we addressed the essential role of Mettl1 in the formation of elongated spermatids and male fertility through m⁷G tRNA modification. We found that ribosome pausing occurs at Pro and Val codons decoded by m⁷G-modified tRNAs in Mettl1-KO testes. Consistent with defective elongated spermatid formation in Mettl1-KO testes, Mettl1 might play an important role in mRNA translation involved in spermatid development, sperm axoneme assembly and sperm individualization (Fig. 7c). During late-stage spermatogenesis, chromatin modifiers cause chromosome compaction to shut down transcription. This stage therefore relies on translation of stored mRNAs, which is regulated by RNA binding proteins^58,59. Our data show that epitranscriptomic regulation of tRNA, m⁷G modification, also plays a critical role during this process. Furthermore, apart from the fertility phenotype, no other developmental defect was observed despite the presence of Mettl1 and m⁷G modified tRNAs in non-gonadal tissues. This indicates that different tissues vary in their susceptibility to abnormal development in response to changes in m⁷G modification.

The finding that Mettl1-KO resulted in decreased expression of the majority, but not all, m⁷G-modified tRNAs (Fig. 5a, b) was consistent with previous studies in mammals and yeast^10,22,27. Critical components of the yeast RTD pathway, Rat1/Dhp1, Met22, and Xrn1, are all present in higher eukaryotes⁶⁰. Therefore, the molecular machinery reported in the yeast RTD pathway may also function in other organisms, although this has not been confirmed in animals. eEF1α1 overexpression partially rescued Mettl1-KO fertility, which implicates a similar system working in animals. Interestingly, our data showed the m⁷G-modified tRNA species to be largely similar between Drosophila and mammals, although there were some differences in Mettl1 dependency on tRNA stability. Although m⁷G-modified tRNAs have a RAGGU motif (Fig. 4f) that is similar to a motif described in a previous study¹⁰, our TRAC-seq analysis showed that tRNA ArgTCT is categorized as m⁷G-modified in testis but not in ovary. This difference might be caused by different read numbers used to calculate cleavage score. Thus, except for ArgTCT, our analysis showed that 22 m7G-modified tRNAs were observed in both testis and ovary (Fig. 4e, Supplementary Fig. 6d), indicating that the repertoire of m7G-modified tRNAs is largely consistent among tissues. Moreover, we found that m⁷G-modified tRNA PheGAA was stable in Drosophila Mettl1-KO (Fig. 5a, b). This is clearly different from that in observed in METTL1-depleted mammalian cells^10,27. Therefore, it would be interesting to conduct a similar analysis in various animals to determine common and different features among m⁷G-modified tRNAs. The RTD in yeast acts on tRNAs lacking one or more of several different modifications⁴⁹; therefore, we speculate that these differences might be caused by varying combinations of other tRNA modifications. Indeed, a subset of hypomodified tRNAs is reduced in Drosophila adult flies lacking cytosine-5 tRNA methyltransferases⁶¹; therefore, it is likely that tRNA decay can occur in diverse cell types, depending upon the combinations of RNA modifications.

It was surprising to find that the only obvious abnormality in Mettl1-KO flies was restricted to fertility because Mettl1 is ubiquitously expressed and m⁷G modification of tRNAs also occurs in nongonadal tissues (Fig. 7e). Notably, there is no post-meiotic transcription in Drosophila testes, which is conserved in mammals⁶². Therefore, proteins involved in spermatid differentiation must be translated from previously produced mRNAs that are stored in a translationally repressed state. During this process, numerous mRNAs are translated and spermatids undergo dynamic morphological changes, which include nuclear condensation and axoneme formation, without supplementation of fundamental translation factors, such as translation elongation factors. Based on our analysis of eEF1α1, we speculate that low eEF1α1 expression may sensitize testes more than other tissues to Mettl1 deficiency. However, we have not reached a definitive conclusion for this important point. It is therefore necessary to understand how hypomodified tRNAs are destabilized in testes. We also showed that Mettl1-dependent m⁷G methylation requires Wh in vitro and that m⁷G modification is lost in wh mutants (Figs. 3a, 4c, Supplementary Figs. 4, 5a–c), indicating that a collaborative role between Mettl1 and Wh is critical for spermatogenesis. However, we do not rule out the possibility that a m⁷G-independent function of Mettl1 or Wh causes part of the observed phenotypes.

Notably, it remains controversial whether METTL1 depletion affects global translation efficiency. METTL1 depletion in human glioblastoma LNZ308 cells²⁷, HuCCT1, and RBE cells²⁶, and in mouse ES cells¹⁰, reduces global translation efficiency. In contrast, METTL1-depletion did not affect overall translation levels in A549, HepG2, and HeLa cells^12,15,28. Considering that initiator tRNA was classified into group 1 tRNA in our analysis, the reduction of initiator tRNA might be presumed to affect global ribosome initiation. However, the abundance of iMet tRNA was only modestly affected in Mettl1-KO flies (Fig. 5b). Therefore, it remains unclear to what extent this change can explain the reduction of TE (Fig. 7a) and the observed phenotypes (Fig. 2) in Mettl1-KO flies. Ribosome profiling data indicate that ribosome collisions occur at codons decoded by group 1 tRNAs (Fig. 6d, e). There appeared to be a weak correlation between codon use of group 1 tRNAs and changes in TE (Fig. 7b); however, that correlation was not supported in codon frequency analysis limited to the Pro codons (Supplementary Fig. 9a). Currently, we do not yet have a clear answer to why we could not detect a correlation between the Pro codon frequency and changes in TE, however, it would be interesting to analyze whether a small number of Pro codons are sufficient to induce ribosome collisions and/or whether the position of Pro codons affects TE.

The GO analysis of cellular component terms of genes that were down-regulated in Mettl1-KO testes revealed that Mettl1 is critical for translation of mRNAs involved in elongated spermatid and mature sperm formation, which is supported by our experimental observations (Fig. 2). Analysis of GO biological process terms of genes that were down-regulated in Mettl1-KO testes revealed that Mettl1 is critical for the translation of mRNAs involved in the catabolism of fructose, which acts as an energy source for spermatozoa⁶³. These data indicate that Mettl1-KO severely impairs protein synthesis from a subset of mRNAs during late rather than early stages of spermatogenesis, resulting in male sterility. We did not find any enrichment or depletion of the conserved m⁷G motif in genes with Mettl1-dependent TE (data not shown), indicating TE changes caused by decreased tRNA abundance rather than direct m⁷G modification of mRNAs are likely to account for the spermatogenesis defects in Mettl1-KO testes.

Interestingly, our ribosome profiling analyses showed increased translation efficiency for 50 transcripts (Fig. 7a). A recent report revealed that internal m⁷G-modified mRNAs with GA-rich motifs are directed to stress granules via Quaking proteins in human cells²⁸. Stress granules contain non-translating mRNAs⁶⁴; therefore, the high TE in Mettl1-KO testes might result from depression of stress granule-stored, m⁷G-modified mRNAs.

Activation of Mettl1 can occur in cancer cells, resulting in stabilization of tRNA (ArgTCT4-1) and increased translation of cell-cycle gene mRNAs, such as CDK6 and others, which promotes tumor progression^26,27. Notably, not only METTL1/WDR4 but also eEF1α1 expression is elevated in hepatocellular carcinoma cell lines and clinical samples⁶⁵, indicating that tRNA stability (ArgTCT4-1) might be synergistically increased in hepatocellular carcinoma cells. Tumor progression depends on the promotion of tRNA (ArgTCT4-1) methylation by METTL1; therefore, modulating tRNA levels in some way might attenuate tumor progression. A combination of drugs targeting the tRNA decay machinery may enhance Mettl1 inhibitor effects.

Methods

Fly stocks

A complete list of fly stocks used in this study is presented in Supplementary Table 2. All stocks were maintained at 25 °C on standard medium. Mettl1-knockout (Mettl1-KO) lines were generated using the transgenic CRISPR-Cas9 method⁶⁶. To generate mutants, we used the following strains: y¹ v¹ nos-phiC31; attP40 host (NIG-FLY stock TBX-0002), y² cho² v¹; Sp hs-hid/CyO (NIG-FLY stock TBX-0009), y¹ w¹¹¹⁸; +; attP2{nos-cas9}⁶⁷, and Df(1)JA27/FM7c, P{w[+mC]=GAL4-Kr.C}DC1, P{w[+mC]=UAS-GFP S65T}DC5, sn[+] (Bloomington Drosophila Stock Center #5193). Disruption of the Mettl1 region in mutants was validated by PCR and Sanger sequencing. All crosses were performed at 25 °C. Oligonucleotide sequences for Mettl1-KO line generation are shown in Supplementary Table 3. To confirm the requirement of Wh in Mettl1-dependent m⁷G methylation, we used the following strain: w* wuho56/FM7c, sn⁺ (Bloomington Drosophila Stock Center #41112). w* / Y; P{Dj-GFP.S}AS1 / CyO (Bloomington Drosophila Stock Center #5417) was used to validate expression of Dj (don juan) in testes. To generate yw; Vasa-GFP and Mettl1^KO1; [Vasa-GFP] males (Supplementary Fig. 9b), we used the following strain: w*; TI{TI}vasEGFP.KI (KYOTO Stock Center 118616).

Fertility assay

To test male or female fertility, a single fly was mated with three control (y¹ w¹¹¹⁸) opposite sex flies at 25 °C for three or 10 days. After mating, the parental flies were removed, and incubation continued for 11 (male) or 4 days (female). Emerged adult flies (progeny) were counted and the average number per vial calculated and evaluated by Tukey’s HSD test. Ten independent vials for each strain were prepared. Each fertility assay was reproduced for at least 2–3 times. The complete list of flies used in these assays is shown in Supplementary Table 2.

Climbing assay

A climbing assay was performed as described previously⁶⁸. Groups of 20 adult male flies (3-days after eclosion) were collected using CO₂ anesthesia and then transferred into a 250 ml graduated glass cylinder. The flies were allowed 120 s to climb past the 200 ml line (17.5 cm from the cylinder bottom). The number of flies above the 200 ml line at 120 sec was counted and the ratio of flies able to climb the cylinder calculated. Five separate assays were performed and subjected to Tukey’s HSD test.

Weight analysis

Groups of 10 adult male flies were weighed on an electronic balance. Three replicates were performed for each strain (y¹w¹¹¹⁸ and Mettl1^KO1). Two-tailed Student’s t-test was performed for statistical analysis.

Courtship assay

All flies were collected within 8 h after eclosion and kept in vials filled with Drosophila standard yeast-based food at 25 °C in 40–60% relative humidity under a 12-h light/dark cycle. Male flies were maintained individually. Virgin females were maintained in groups of 10–30 flies. y¹w¹¹¹⁸ females were used as mating partners for male flies. Males and females 5–8 days after eclosion were used for video recordings. All video recordings were performed during the light phase of the light/dark cycle. Video annotation software BORIS was used for analysis⁶⁹.

To quantify the copulation rate, a pair of male and female flies were introduced into each artificial chamber (diameter 10 mm, height 4 mm, bottom coated with Drosophila standard food), and video recording was performed using a CMOS camera (DFK 33UP1300, The Imaging Source Asia Co., Ltd) equipped with a zoom lens (M0814-MP2, CBC Optics Co., Ltd.) for 24 h at one fps. y¹w¹¹¹⁸, Mettl1^KO1, or tut-KO males were used to serve as the test flies. We calculated the copulation rate by determining whether each pair had copulated or not in 24 hours.

Measurement of adult progeny emerging rate

Test crosses were performed using heterozygous female (Mettl1^KO1/FM7 or y¹w¹¹¹⁸/FM7) and wild-type male flies. One virgin female and three males were crossed and removed 5 days after mating. Emerged offspring were counted 14 days after mating and then evaluated by two-tailed Student’s t-test.

Western blotting

Western blotting was performed as described previously⁷⁰. Ovaries were collected from 3–4-day-old adult females. An anti-Mettl1 monoclonal antibody, an anti-Wh monoclonal antibody (both generated in this study), and an anti-Tubulin antibody [E7 culture supernatant, Developmental Studies Hybridoma Bank] were used as primary antibodies. A goat anti-mouse IgG (H + L) Poly-HRP secondary antibody (Thermo Fisher, USA, #32230, 1:5000–10000) was used as secondary antibody.

Measurement of ovary size

Ovaries were dissected from 7-day-old adult females. Ovaries were fixed in 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, Japan) for 15 min at room temperature or overnight at 4 °C. After washing with PBS-T three times, ovary size was measured using a stereomicroscope and cellSens (Olympus, Japan) imaging software. Significant differences in ovary size were evaluated by Tukey’s HSD test.

Immunofluorescence

Immunostaining analysis was conducted as described previously⁷¹. Testes from 3-day-old adult males and ovaries from 7-day-old adult females were dissected in ice-cold PBS. Tissues were fixed in 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque #09154-85) for 15 min at room temperature. The fixed tissues were washed five times with PBS containing 0.1% Tween 20 (PBS-T), and then incubated twice in PBS-T containing 0.2% Triton X-100 and 0.1% BSA (PBS-BT) for 10 min. The washed tissues were then incubated with primary antibodies in PBS-BT at 4 °C for 1 day. Primary antibodies used were: anti-Aub (4D10)⁷², anti-acetylated tubulin (Sigma, 1:100 dilution), anti-boule (1:250 dilution, a kind gift from S. Wasserman, University of California San Diego), anti-FLAG (M2, Sigma, 1:1000 dilution; to detect Mettl1-3×Flag), anti-Fas3 (7G10, Developmental Studies Hybridoma Bank), and anti-Tj (10009Ab-1, NIG-FLY, Japan). Incubated tissues were then washed five times and further washed twice with PBS-BT. Next, the tissues were incubated at 4 °C for 1 day with the following secondary antibodies as appropriate: Alexa 546-conjugated anti-mouse IgG (Thermo Fisher, A11030, 1:250 dilution), Alexa 488-conjugated anti-mouse IgG (Thermo Fisher, A11029, 1:250 dilution), Alexa 488-conjugated anti-rabbit IgG (Thermo Fisher, A11008, 1:250–1000 dilution), and Alexa 546-conjugated anti-rabbit IgG (Thermo Fisher, A11071, 1:250–1000 dilution). After washing three times for 10 min in PBS-T, tissues were mounted in Vectashield Mounting Medium containing DAPI (Vector Laboratories). Images of testes and ovaries were taken using a confocal microscope (Zeiss LSM900). 405 nm or 488 nm laser were used for excitation. 400–505 nm and 505–700 nm filters were used for fluorescence collection. Seminal vesicles and elongated spermatids in testes were fixed by the same method and observed by light field confocal microscopy (Zeiss LSM900). Using independent fluorescence channels, we validated that the green signals in Fig. 2b, d, f did not come from autofluorescence of the Drosophila tissue (data not shown).

Production of monoclonal antibodies

Recombinant full-length Drosophila Mettl1 protein tagged with maltose binding protein and Wh (N-terminal 200 amino acids) tagged with glutathione S-transferase (GST) were prepared as antigens and purified from E. coli. Mice were immunized with each antigen. The production and selection of hybridomas were performed as previously described⁷³.

Plasmid construction

To produce plasmids expressing Drosophila Mettl1, Mettl1-Catalytic dead (Cd), Mettl1-3×Flag, nosp-Mettl1, UASt-Mettl1, and UASt-eEF1α1, Mettl1 and eEF1α1 coding regions were amplified using KOD plus DNA polymerase (Toyobo #KOD-201). The Nanos promoter region was amplified from pBFv-nosp-Cas9. The amplified fragments and vector pBFv-UAS3 were fused using the In-Fusion® HD Cloning Kit (TaKaRa). The oligonucleotide sequences used are shown in Supplementary Table 2. To produce the plasmids expressing Mettl1 and Wh in E. coli, Mettl1 and Wh coding sequences were amplified using KOD plus DNA polymerase (Toyobo) and were then integrated into pGEX 5x-1 (Amersham), or pMAL (NEB). The oligonucleotide sequences used are shown in Supplementary Table 3.

GST pull-down assays

GST pull-down assays were performed as previously described⁷⁴. Purified GST-tagged proteins were incubated in methylation buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.2 mM DTT, 10 mM EDTA, 0.2 mM MgCl₂) with Glutathione Sepharose™ 4B (GE healthcare 17-0756-01) for 1 h at 4 °C to immobilize GST-tagged proteins. Immobilized proteins were washed three times with methylation buffer. After washing, MBP or MBP-tagged protein was added and incubated for 4 h at 4 °C. After incubation, proteins were washed five times with methylation buffer, and then eluted with 2×Sample buffer [100 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 200 mM DTT]. Samples were incubated for 5 min at 95 °C, and were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were detected by Coomassie brilliant blue staining.

Preparation of protein samples for LC-MS/MS analysis

To prepare immunoprecipitated samples for LC-MS/MS, the anti-3×FLAG antibody used for immunoprecipitation was cross-linked to beads using dimethyl pimelimidate (Thermo Fisher). Immunoprecipitation was performed using mouse anti-FLAG M2 or mouse non-immune IgG (negative control) and ovaries from negative control (Oregon R) or 3×Flag-tagged Mettl1 (attp40[Mettl1-Flag]) flies. We used two different buffer conditions: Tris-Triton buffer [20 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl₂, 2 mM DTT, 0.1% TritonX-100] and Hepes-NP40 buffer [30 mM Hepes-KOH (pH 7.3), 150 mM KOAc, 5 mM MgOAc, 5 mM DTT, 0.1% NP40]. LC-MS/MS analysis was performed as described previously⁷⁵. The immunoprecipitated proteins were separated on 4–12% SDS-PAGE gels and stained with SimplyBlue (Thermo Fisher). Gel containing proteins was excised and cut into pieces of approximately 1 mm². Proteins in the gel pieces were reduced with DTT (Thermo Fisher), alkylated with iodoacetamide (Thermo Fisher), and digested with trypsin and lysyl endopeptidase (Promega) in a buffer containing 40 mM ammonium bicarbonate, pH 8.0, overnight at 37 °C. The resultant peptides were analyzed on an Advance UHPLC system (AMR/Michrom Bioscience) connected to a Q Exactive mass spectrometer (Thermo Fisher). The raw mass spectrum was processed using Xcalibur (Thermo Fisher). The raw LC-MS/MS data were analyzed against the UniprotKB database restricted to Drosophila melanogaster using Proteome Discoverer version 1.4 (Thermo Fisher) with the Mascot search engine version 2.5 (Matrix Science). A decoy database comprising either randomized or reversed sequences in the target database was used for false discovery rate (FDR) estimation, and Percolator was used to evaluate false positives. Search results were filtered against 1% global FDR for high confidence level. Quantitative analyses were performed using scaffold 5 (Proteome Software Inc, Portland, OR), and volcano plots of identified proteins were generated using Student’s t-test or Fisher’s exact test under a normalization scheme. Single replicate was analyzed following to standards in the field.

Northern blotting

Northern blotting was performed as described previously⁷⁶. Sample RNAs were denatured with Gel loading buffer II (Invitrogen #AM8546G) for 3 min at 65 °C. Sample RNAs were resolved by electrophoresis on 12% urea-PAGE gels, which contain 7 M urea, 10× TBE (1 M Tris base, 1 M boric acid, 0.02 M EDTA), and 12% acrylamide. To assess m⁷G site-specific cleavage of tRNAs and to confirm tRNA levels, 1.25 μg (from ovaries) and 0.5 μg (from testes) sample RNAs were resolved by electrophoresis and then transferred to Hybond N+ membranes (Cytiva). Transferred RNAs were UV-crosslinked with 1200 × 100 mJ/cm2 irradiance at 254 nm. After crosslinking, hybridization was conducted at 42 °C overnight in 7% SDS, 0.2 M sodium phosphate (pH 7.2), and 1 mM EDTA with ³²P-labeled DNA probes. Membranes were then washed with 2× saline sodium citrate containing 0.1% SDS at 42 °C. To detect in vitro m⁷G methylation, the hybridization step was skipped. The oligonucleotide sequences used are shown in Supplementary Table 3.

In vitro methylation assay

GST, GST-Wh, MBP, MBP-Mettl1, and MBP-Mettl1-Cd were produced and purified from E. coli. An in vitro tRNA methylation assay was performed essentially as described previously with some modifications⁷⁷. Purified GST and MBP protein (1 μg each) were incubated with chemically synthesized RNA (0.3 nmol) at 26 °C in a buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl₂, 2 mM DTT and 2 μCi/mL S-adenosyl-L-[methyl-¹⁴C] methionine (PerkinElmer) for 3 h. RNAs were then isolated with phenol:chloroform and ethanol precipitated. The resultant RNAs were separated on 7 M urea-containing denaturing polyacrylamide gels. The gels were stained with Toluidine Blue O in 0.5 × TBE, destained with 0.5 × TBE, and dried. The ¹⁴C-labeled RNA bands were visualized on a Typhoon FLA900 (GE Healthcare).

m⁷G site-specific reduction and cleavage

Total RNA from ovaries and testes were isolated using Isogen (Nippon Gene, 311-02501) following the manufacturer’s instructions. Ten micrograms of total RNA were incubated with 0.1 M NaBH₄ and 1 mM free m⁷GTPfor 30 min on ice in the dark. Reduced RNAs were precipitated with 3 M sodium acetate pH 5.2 (Thermo Fisher), glycogen (Nacalai Tesque) and ethanol at −20 °C for at least 1 h. RNA samples reduced by NaBH₄ were then reacted with aniline-acetate solution (H₂O:glacial acetate acid:aniline, 7:3:1) at room temperature in the dark for 2 h to cause m⁷G site-specific cleavage^46,47. The cleaved RNAs were detected by northern blotting. Total RNA from Wh-KO males was isolated from 3-day-old adult male testes of w* wh56/FM7c sn+ flies (Bloomington Drosophila Stock Center #5417).

TRAC-seq and profiling of tRNA expression

Sequencing and data analysis were performed as previously described⁴⁸ with some modifications. Small RNAs (<200 nt) were isolated from whole ovary (one sample) and testis (three replicates) RNAs using a mirVana miRNA Isolation Kit (Thermo Fisher, AM1561) following the manufacturer’s instructions. To exclude methylation inhibiting the reverse transcription of tRNAs, 2.5 μg of extracted small RNAs were treated with recombinant ALKB and ALKB D135S proteins. Demethylated small RNAs were reduced and cleaved by the method described in the “m⁷G site-specific reduction and cleavage” section of the manufacturer’s protocol. After cleavage and ethanol precipitation, small RNA sequencing libraries of m⁷G tRNAs were constructed using a NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S). In preparing the library, TGI-RT (Thermostable Group II intron reverse transcriptase, TGIRT-III-10, InGex) was used instead of the reverse transcriptase in the library kit. The prepared libraries were sequenced on a Novaseq 6000 system. The 290 reference sequences of mature Drosophila tRNAs (i.e., dm6-mature-tRNAs) were downloaded from GtRNAdb⁷⁸. Identical sequences were removed to yield 84 unique sequences and a “CCA” sequence was added to the 3′-end of each tRNA (i.e., “dm6-mature-tRNAs_unique_CCA”). The 5′- and 3′-adapter sequences of the sequenced reads were trimmed and low-quality sequences were filtered using FASTP ver. 0.23.2⁷⁹ to obtain “clean reads”. The clean reads were mapped to the reference “dm6-mature-tRNAs_unique_CCA” using BOWTIE ver. 1.3.1⁸⁰. The number of reads mapped on each tRNA (i.e., “TotalReads”) was obtained from the mapping data (bam) using PICARD ver. 3.0.0 (https://broadinstitute.github.io/picard/command-line-overview.html). The number of reads starting at each site of each tRNA (i.e., “StartReads”) was obtained using the custom shell script. The ratio between StartReads and TotalReads as an indicator of the m⁷G-dependent read cleavage (i.e., “StartRatio”) was calculated for each site i of each tRNA of chemically-treated and non-treated samples as:

$${StartRati}{o}_{i}={StartRead}{s}_{i}/{TotalRead}{s}_{i}$$

(1)

The cleavage score as an overall indicator of the m⁷G modification (i.e., “CleavageScore”) was calculated for each site i of each tRNA of WT and Mettl1-KO samples as:

$${CleavageScor}{e}_{i,{WT\; or\; Mettl}1-{KO}}=\log 2\left(\frac{{StartRati}{o}_{{treat}}}{{StartRati}{o}_{{no}{n}_{{treat}}}}\right)$$

(2)

The difference in cleavage score between WT and Mettl1-KO samples (i.e., “Cleavage score”) was defined as another indicator of Mettl1-dependent m⁷G modification as:

$${Cleavage\; scor}{e}_{i}={CleavageScor}{e}_{i,{WT}}-{CleavageScor}{e}_{i,{Mettl}1}$$

(3)

For testes, these feature values were averaged from the data of three replicates. The sites with StartRatio_WT,treat ≥ 0.02 were regarded as candidates. Those with CleavageScore_i,WT ≥ 2 and Cleavage score_i ≥ 2 were extracted as the final candidate m⁷G-dependent cleavage sites. The sequences of ± 10 bases around the candidate m⁷G site were analyzed to detect the conserved motif for the m⁷G modification using MEME ver. 5.5.2⁸¹.

The chemically non-treated testis samples for TRAC-seq (WT: three replicates; Mettl1-KO: three replicates) were processed to profile tRNA expression using the tRAX pipeline⁸².

Acid urea PAGE

Acid urea PAGE was performed as previously described⁸³. Briefly, total RNAs were treated with 0.1 M Tris pH 9.0 at 42 °C for 1 hr. Treated and control RNA samples were then separated by acid urea PAGE.

Ribosome footprinting and RNA-Seq

Cell lysates were prepared as previously reported with modifications⁸⁴. Testes or male body (with testis removed) were dissected from 2–3-day-old adult males in ice-cold PBS containing 100 µg/ml cycloheximide. Dissected testes were immediately frozen in liquid nitrogen and stored at −80 °C. We prepared 600 pairs of testes per sample and two replicates from wild-type (yw) and Mettl1-KO (Mettl1^KO1) males, respectively. Male bodies were prepared from 100 male adults for each strain. Frozen testes or body samples were mixed with 300 µl of frozen lysis buffer droplets (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 1% Triton X-100, 100 µg/ml chloramphenicol, and 100 µg/ml cycloheximide), and then ground at 3000 rpm for 15 s in a Multi-beads Shocker (YASUI KIKAI). The lysate was slowly thawed at 4 °C and treated with 10 U Turbo DNase (Thermo Fisher) on ice for 10 min to remove genome DNA. The supernatant was further centrifuged at 20,000 g for 30 min at 4 °C.

Library construction of ribosome profiling (or Thor-Ribo-Seq) was conducted as described previously^54,84. Cell lysate containing 8.5 µg total RNA was treated with 20 U RNase I (Epicenter) in a 300 µl reaction at 25 °C for 45 min. Then, the reaction was stopped with 200 U of SUPERase•In RNase Inhibitor (Thermo Fisher). Ribosomes were isolated by sucrose cushion ultracentrifugation at 100,000 rpm at 4 °C for 1 h using a TLA110 rotor (Beckman Coulter). RNAs in the ribosome pellet were isolated with TRIzol-LS (Thermo Fisher) and a Direct-zol RNA MicroPrep kit (Zymo Research). Purified RNA fragments ranging from 17 to 34 nt were selected by gel electrophoresis, dephosphorylated by T4 polynucleotide kinase (New England Biolabs), and ligated to preadenylated linkers containing the T7 promoter region using T4 RNA ligase 2, truncated KQ (New England Biolabs). Before ligation, the linkers were preadenylated by Mth RNA Ligase (NEW England Biolabs). Linker-ligated RNAs were treated with riboPOOL for D. melanogaster (siTOOLs Biotech) to remove fragments of ribosomal RNAs. In vitro transcription was then performed using a T7-Scribe Standard RNA IVT Kit (CELLSCRIPT). RNAs were dephosphorylated by T4 polynucleotide kinase (New England Biolabs) and ligated with linker for reverse transcription. The cDNAs were synthesized by ProtoScript II (New England Biolabs) and amplified using Phusion High-Fidelity DNA Polymerase. The DNA libraries were sequenced on the HiSeq X platform (Illumina) with the paired-end 150-bp option.

For RNA-Seq, we extracted total RNA from the lysate originally used for ribosome profiling using TRIzol LS (Thermo Fisher) and a Direct-zol RNA MicroPrep Kit (Zymo Research). Sequencing libraries were prepared using 1 µg of the total RNA and the SEQuoia Express Standard RNA Library Prep Kit (Bio-Rad). rRNA depletion was performed with riboPOOL for D. melanogaster (siTOOLs Biotech). Sequence data were processed using previously established methods^85,86. Translation efficiency change was determined using the DESeq2 package⁸⁷. Gene ontology (GO) analysis was performed with DAVID^88,89.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data supporting the findings of this study are available from the corresponding authors upon request. The original raw data in this study are publicly available. The mass analyses data generated in this study have been deposited in ProteomeXchange (PXD044501) / jPOST repository (JPST002287). The TRAC-Seq data have been deposited in the DNA Data Bank of Japan (DDBJ) (PRJDB16512). The ribosome profiling, and RNA-Seq data obtained in this study are deposited in the National Center for Biotechnology Information (NCBI) database (GSE241519). Source data are provided with this paper.

Code availability

The codes for data analysis of TRAC-seq and tRNA expression are available in Zenodo (https://doi.org/10.5281/zenodo.13340795)⁹⁰. Source codes of softwares for ribosome profiling analysis were shared at GitHub (https://github.com/ingolia-lab/RiboSeq). The codes deposited in Zenodo (https://doi.org/10.5281/zenodo.7477706)⁹¹ were used for data analyses of ribosome footprinting.

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Acknowledgements

We thank T. Suzuki for critical suggestions on this project, A. Terui, A. Masuda, Y. Kitahara, Y. Nagashima, and T. Miyoshi for technical assistance, Y. Shichino and M. Mito for technical advice in ribosome footprinting, and M. Masukawa and S. Kobayashi of the Drosophila Genetic Resource Center in Kyoto Institute of Technology and the Bloomington stock center, respectively, for providing the fly strains. We also Jeremy Allen, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work was partially supported by JSPS KAKENHI under grant numbers JP18H02379, JP16H06279 (both PAGS), JP23H02415 (SI) and JP22H02669 (KS), the Takeda Science Foundation, the Yamada Science Foundation, the Mochida Memorial Foundation (KS), The Naito Science & Engineering Foundation and the Uehara Memorial Foundation (KS), AMED under grant number JP20gm1410001 (SI), and the Program of the Joint Usage/Research Center for Developmental Medicine and High Depth Omics, IMEG, Kumamoto University (KS). K.T. was a recipient of the RIKEN Student Researcher program and a World-leading Innovative Graduate Study Program in Proactive Environmental Studies (WINGS-PES) from The University of Tokyo. This study used the HOKUSAI SailingShip supercomputer facility at RIKEN.

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Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
Shunya Kaneko, Keita Miyoshi & Kuniaki Saito
Graduate Institute for Advanced Studies, SOKENDAI, Shizuoka, Japan
Shunya Kaneko, Keita Miyoshi & Kuniaki Saito
RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Saitama, Japan
Kotaro Tomuro & Shintaro Iwasaki
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
Kotaro Tomuro & Shintaro Iwasaki
Center for Genome Informatics, Joint Support-Center for Data Science Research, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
Makoto Terauchi & Hideki Noguchi
Graduate School of Science, Nagoya University, Nagoya, Aichi, Japan
Ryoya Tanaka & Azusa Kamikouchi
Institute for Advanced Research, Nagoya University, Nagoya, Aichi, Japan
Ryoya Tanaka & Azusa Kamikouchi
Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
Shu Kondo
Liaison Laboratory Research Promotion Center, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Kumamoto, Japan
Naoki Tani
Department of Chromosome Biology, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University, Kumamoto, Japan
Kei-Ichiro Ishiguro
Department of Genomics and Evolutionary Biology, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
Atsushi Toyoda
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Aichi, Japan
Azusa Kamikouchi

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Shunya Kaneko
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Contributions

Shunya Kaneko: conceptualization, formal analysis, investigation, methodology, visualization, validation, writing-original draft, writing-review and editing. Keita Miyoshi: investigation; methodology; visualization; validation, writing-review and editing. Kotaro Tomuro: data curation, investigation; methodology; writing-review and editing. Makoto Terauchi: data curation, software; investigation; methodology; writing-original draft; writing-review and editing. Ryoya Tanaka: investigation, methodology and editing. Shu Kondo: resource; methodology and editing. Naoki Tani: LC/MS/MS; methodology and editing. Kei-Ichiro Ishiguro: Investigation; methodology and editing. Atsushi Toyoda: methodology. Azusa Kamikouchi: investigation, methodology and writing-review and editing. Hideki Noguchi: data curation, investigation; methodology; writing-original draft; writing-review and editing. Shintaro Iwasaki: data curation, investigation; methodology; writing-original draft; writing-review and editing. Kuniaki Saito: conceptualization; funding acquisition; investigation; methodology; project administration; supervision; writing-original draft; writing-review and editing.

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Kaneko, S., Miyoshi, K., Tomuro, K. et al. Mettl1-dependent m⁷G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster. Nat Commun 15, 8147 (2024). https://doi.org/10.1038/s41467-024-52389-0

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Received: 01 September 2023
Accepted: 03 September 2024
Published: 24 September 2024
Version of record: 24 September 2024
DOI: https://doi.org/10.1038/s41467-024-52389-0

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