N7-methylguanosine methylation of tRNAs regulates survival to stress in cancer

Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5’ tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.


INTRODUCTION
The ability of cancer cells to survive stress is crucial for tumour to progress and adapt to therapy, emphasising the need to identify targetable molecular pathways challenging cancer cell survival.RNA chemical modifications are emerging as an additional layer of biological information that dynamically regulate cellular functions, including self-renewal, proliferation, or survival to stress [1,2].Although the study of RNA modifications dates back to the 1950s, investigations into RNA modifications in the context of human cancer, began only around the 1990s.This focused exploration of RNA modifications in human cancer has revealed valuable insights into their role in tumorigenesis [3][4][5][6][7][8][9].Understanding the specific involvement of RNA modifications in human cancer holds great promise for unravelling novel molecular mechanisms and identifying potential targets for diagnosis and treatment.
Over 170 RNA chemical modifications constitute the epitranscriptome, and their function is currently being investigated [10].RNA modifications are primarily found in transfer RNA (tRNAs), where they regulate tRNA stability, folding, and protein synthesis [11].Dysregulation of tRNA-modifying enzymes is frequently associated with stress-related diseases, including cancer [12].For instance, reduced expression of TYW2 enhances migration in colorectal cancer [13].Furthermore, altered tRNA modifications are implicated in cancer chemoresistance [14][15][16][17].Notably, loss of tRNA cytosine-5 methylation (m 5 C) leads to increased production of stress-induced tRNA fragments (tRFs), sensitising cancer cells to stress and chemotherapy [18,19].These observations underscore the critical role of tRNAs and tRNA modifications in stress response, suggesting a potential link between tRNA modifications and therapy resistance.However, the precise molecular mechanisms that connect the stress response pathway to tRNA modifications remain largely unknown.
Here, through transcriptome-wide approaches to map m 7 G, functional assays, and Mettl1 knockout mouse models, we provide compelling evidence that m 7 G is essential for protecting tRNAs from stress-induced cleavage and protein synthesis regulation.Depletion of METTL1 sensitises cancer cells to stress, resulting in heightened cytotoxic responses to conventional cancer treatments both in vitro and in vivo.Our study unveils the significance of m 7 G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to enhance the sensitivity of cancer cells to therapy.

RESULTS
Loss of N 7 -methylguanosine in tRNAs increases stress-induced tRNA cleavage Hypomodified tRNAs have been linked with stress responses and chemosensitivity to targeted therapies [12,[14][15][16][17].Because tumour cell tRNAs have been shown to be methylated by METTL1 [20,31,32], we postulate a potential connection between the loss of m 7 G deposition and stress signalling.
To validate the methylation of tRNAs by METTL1 in prostate cancer (PCa) cells (PC3), we developed a high-throughput method that precisely maps m 7 G deposition in RNA.This method utilises the sensitivity of methylated guanosines to chemical treatments, followed by RNA sequencing (RNA-seq) (Supplementary Fig. 1D) [33].The absence of METTL1-mediated m 7 G methylation was confirmed in METTL1 knock-out (METTL1-KO) cells (Supplementary Fig. S1A-C).RNA-seq was performed on treated and untreated RNAs (Supplementary Fig. S1D), and the cleavage sites in the treated RNAs served as a surrogate for m 7 G deposition sites.
Because loss of m 5 C at the variable loop of tRNAs has been associated with an increased generation of stress-induced 5'tRFs processed by angiogenin [14,18,19,34], our next objective was to determine whether the loss of m 7 G could similarly impact on tRNA processing.We found no significant differences in the stability of mature tRNA in PC3 cells lacking METTL1 (Supplementary Fig. S1G).Subsequently, we examined the generation of oxidative stress-induced 5′tRFs.As expected, tRNAs were cleaved in PC3 WT cells (Fig. 1C, D), resulting in two different 5′tRFs: longer fragments presumably corresponding to 5′tRNA halves cleaved at the anticodon loop, and shorter fragments presumably cleaved at the D loop.Fragment production peaked at 2 h after stress stimulation and disappeared at 8 h, in accordance with previous reports [14].Interestingly, the production of tRFs in METTL1-KO cells was higher at later points, suggesting a prolonged stress response (Fig. 1C, D).Furthermore, tRNA cleavage in PC3 METTL1-KO cells was not suppressed in the presence of the angiogenin inhibitor, indicating the involvement of another endonuclease in tRNA cleavage (Fig. 1E, F, Supplementary Fig. S1H).
Thus, our data demonstrate that the biogenesis of distinct stress-induced 5'tRFs is driven by METTL1-specific methylation.

Loss of N 7 -methylguanosine in tRNAs disrupts proteostasis
Stress-mediated tRNA cleavage and 5′tRFs are important components of the cellular stress response that ultimately inhibits global protein synthesis to allow cells to recover from stress and survive [35].Accordingly, analysis of protein synthesis rate in PC3 METTL1-KO cells showed a significative reduction in protein translation and a weakened recovery of protein synthesis rates compared to WT cells (Fig. 1G, Supplementary Fig. S1I).These findings were validated in the PCa cell line DU145 using doxycycline-inducible shRNAs against METTL1.Consistently, the immediate loss of m 7 G deposition in tRNAs led to a consistent decrease in protein synthesis (Supplementary Fig. S1J, K).These data strongly indicated that loss of m 7 G caused stress-induced repression of protein synthesis across different cell lines.To further asses proteostatic stress in the absence of METTL1, we examined the formation of protein aggregates.METTL1-deficiency led to a significant increase in protein aggregates formation, confirming an unbalanced protein homoeostasis (Fig. 1H).
In summary, we conclude that loss of METTL1-mediated tRNA methylation disturbs tRNA processing and leads to unbalanced proteostasis.
Loss of METTL1 disrupts the autophagic flux Since tRNA modifications and tRFs can modulate translational programmes, we next used proteome-wide profiling of METTL1inducibly silenced DU145 cells to specifically analyse the direct proteome changes caused upon the removal of METTL1.We identified subtle expression changes in common proteins upon METTL1-silencing (Supplementary Table S2).GO term analysis revealed enrichment in catabolic processes such as proteolysis, peptidase and hydrolase activities (Fig. 2A), suggesting a potential link between METTL1 and autophagy in line with previous findings [36].To explore this, we examined the levels of LC3 II and p62 in PC3-WT and METTL1-KO cells.No significant expression changes were found in METTL1-KO cells compared to WT cells growing in culture conditions with complete or reduced medium with amino acids and serum (Fig. 2B, C, Supplementary Fig. S2A-C).However, METTL1 depletion in PC3 cells significantly increased the levels of LC3 II after chloroquine (CQ) treatment, which inhibits the latest steps of autophagy, indicating that the loss of METTL1 enhanced autophagosome formation (Fig. 2B, Supplementary Fig. S2A).p62 levels remained unchanged after CQ treatment, most likely due to reduced autophagosome resolution (Fig. 2B, Supplementary Fig. S2A).Amino acids and serum starvation in PC3 METTL1-KO cells led to higher LC3 II accumulation, which was further increased by CQ, suggesting impaired fusion of autophagosomes with lysosomes in these cells (Supplementary Fig. S2C).Similar effects were observed in DU145 METTL1-KO cells (Supplementary Fig. S2D, E). p62 levels were scarcely changed after 4 h of starvation in PC3 and DU145 METTL1-KO cells (Fig. 2C, Supplementary Fig. S2B, C, D, E).However, autophagy induction with rapamycin treatment significantly reduced p62 levels in WT cells but failed to stimulate p62 degradation in METTL1-KO cells, indicating that METTL1 is necessary for autolysosome maturation (Fig. 2C, Supplementary Fig. S2B).
To confirm impaired autolysosome maturation in METTL1-deficient cells, we utilised U2OS cells expressing the autophagosome reporter LC3-GFP.Silencing of METTL1 using siRNAs induced an increased accumulation of GFP+ puncta, confirming an increased formation of autophagosome vesicles (Fig. 2D, Supplementary Fig. S2F).Additionally, PC3-WT and METTL1-KO cells were transfected with LC3-GFP reporter plasmid and accumulation of LC3-GFP + /lysotracker+ or LAMP1 + -vesicles was measured.In PC3-WT cells, rapamycin induction resulted in accumulation of autolysosomes, indicating an increased autophagic flux.However, in METTL1-KO cells, the formation of autolysosomes did not change compared to rapamycin-untreated cells, suggesting reduced autophagic flux (Fig. 2E, F, Supplementary Fig. S2G, H, I).Thus, our data indicated that METTL1 loss disrupted the autophagic flux by impairing or reducing autophagy resolution.Although Han et al. [26] observed a decreased translation of mTOR and other negative autophagy regulators in oesophagus squamous cell carcinoma, we did not find significant or consistent changes in phosphorylation of downstream effectors of mTORC1 in PCa cell lines (Supplementary Fig. S2J).
Together, our data revealed that METTL1 is necessary to resolve autophagy independent of mTOR status, its loss induces unresolved autophagy and increased proteostatic stress.
Inhibiting the expression of METTL1 leads to a decrease in cell viability tRNA modifications and tRFs play a critical role in cell viability and survival under stress conditions and can act as stress sensors [14,18,19,35,37,38].Taken together, these findings suggest that the loss of METTL1 could reduce cell viability.Indeed, our functional analysis demonstrated that the loss or knockdown of METTL1 in PCa cells decreased cell proliferation and reduced 3D growth (Fig. 3A, B, Supplementary Fig. S3A).Moreover, METTL1-deficient cells exhibited increased cell sensitivity to oxidative stress as shown by a decrease in IC 50 to H 2 O 2 (Supplementary Fig. S3B).Consequently, METTL1 loss resulted in decreased cell viability in response to oxidative stress or UV exposure (Supplementary Fig. S3C, D).Considering that autophagy resolution was disrupted in METTL1-deficient cells, we investigated whether autophagy induction could further compromise cell survival.As expected, PC3 METTL1-KO cells exhibited increased sensitivity to rapamycin compared to WT cells (Fig. 3C, Supplementary Fig. S3E), likely due to the accumulation of unresolved autophagosomes.The survival defects observed in PC3 METTL1-KO cells were rescued upon re-expression of a wild-type METTL1 form, but not by a catalytically inactive version (AFPA), indicating that tRNA methylation is necessary for survival regulation (Fig. 3D, Supplementary Fig. S3F).Interestingly, PC3 METTL1-KO cells exhibited higher tolerance than WT cells to 3-methyladenine (3MA), which blocks autophagosome formation by inhibiting class III PI3K [39].This suggested that reducing autophagy initiation could partially restore METTL1-KO cells viability (Fig. 3E).The re-expression of wild-type METTL1 significantly reduced cell viability, while the METTL1 inactive mutant partially reduced cell viability (Fig. 3F).

METTL1 inhibition sensitises cells to genotoxic stress
Disruption of autophagy frequently leads to the accumulation of reactive oxygen species (ROS), which is often associated with heightened genotoxic stress [43,44].We next examined whether the dysregulation of autophagy in the absence of METTL1 affected the cellular redox balance.We detected higher ROS production in PC3 METTL1-KO than in WT cells (Fig. 4A, Supplementary Fig. S4A).Furthermore, METTL1 deletion significantly increased the formation of γH2AX nuclear foci, indicative of DNA double-strand breaks, accompanied by a significant increase in the formation of BRCA1 foci near the sites of DNA damage [45] (Fig. 4B, C, Supplementary Fig. S4B, C).
Because autophagy and senescence function as survival mechanisms to resolve stress conditions [44,48], we investigated whether inhibiting METTL1 would sensitise cells to chemotherapy-induced stress.METTL1 deletion in PC3 cells increased cellular sensitivity to chemotherapeutic agents like Docetaxel and Etoposide (Fig. 4E, F).Re-expression of wild-type METTL1, but not the catalytically inactive mutant AFPA, in PC3 METTL1-KO cells reduced their sensitivity to genotoxic stress (Fig. 4G), highlighting the importance of METTL1-dependent methylation in cellular stress responses.
In summary, our findings suggest that inhibiting METTL1 alone or in combination with chemotherapeutic agents could enhance the elimination of cancer cells within a tumour by rendering them more susceptible to genotoxic stress and increasing their responsiveness to chemotherapy.
Immunohistochemical analysis confirmed high expression of METTL1 in luminal (K18 +) cells in human prostatic carcinoma samples (Fig. 5D).To further investigate METTL1 expression in selfrenewing cells of human cancer, we employed an in vitro model utilising formation by cancer stem cells.Tumoursphere cultures derived from PC3 and DU145 cells exhibited higher expression of METTL1, along with the stem-cell markers, compared to cells in 2Dgrowing conditions, confirming higher expression of METTL1 in self-renewing human PCa cells (Fig. 5E, Supplementary Fig. S5B).
In summary, our findings indicate that METTL1 is prominently expressed in luminal cells, which are frequently identified as the cell of origin in mouse and human PCa.

Inhibition of Mettl1 expression sensitises cancer cells to chemotherapy in vivo
To investigate the impact of inhibiting Mettl1 in an in vivo setting, we conditionally deleted Mettl1 in the prostatic epithelium of PtencKO mice (PtencKO/Mettl1 flox/flox ) (Fig. 6A, Supplementary Fig. S6A).Conditional Mettl1 loss led to increased expression of autophagy and genotoxic stress markers in cancer cells (Fig. 6A, B).Accordingly, Mettl1-deficient tumours exhibited a significant increase in apoptosis (Fig. 6A, B), leading to a substantial reduction in tumour mass (Fig. 6C).Histological examination revealed that WT tumours were invasive, composed of proliferative (Ki67 +) luminal (K8 +) and basal (K14 +) cells.In contrast, Mettl1-deficient tumours were encapsulated and predominantly comprised of luminal cells with low proliferative potential (Fig. 6D, E).Furthermore, cytometry analyses indicated that depletion of Mettl1 resulted in a cell subpopulation distribution similar to non-tumoural tissue (Fig. 6F, G).These data suggest that cancer cells and cancer stem-like cells are sensitive to the absence of this enzyme.
To test whether the stress-activated programme in Mettl1 -/- cancer cells could alter their sensitivity to chemotherapy in vivo, we administered Docetaxel, commonly used for treating of metastatic castration-resistant prostate cancer (mCRPC) [56], to mice-bearing tumours.While Docetaxel treatment induced a modest growth reduction in wild-type tumours (Fig. 7A), it significantly reversed the growth and reduced the size of METTL1-KO tumours, almost to the initial stages of tumour formation (Fig. 7B).Docetaxel-treated METTL1-KO tumour cells exhibited dysregulated autophagy, increased DNA damage and apoptosis, leading to a reduction in cells re-entering the cell cycle (Ki67+cells) compared to wild-type cells (Fig. 7C, D).Similar results were obtained using another genotoxic agent, Etoposide (Supplementary Fig. S7).Thus, our data demonstrate that METTL1-deficient tumours fail to activate survival pathways in response to stress.
Taken together, our findings suggest that METTL1 plays a role in regulating cancer cell stress responses.Its inhibition results in increased sensitivity to stress, decreased cell viability, and reduced survival.Consequently, the inhibition of METTL1, alone or in combination with chemotherapeutic options, leads to a substantial reduction in tumour mass, resulting in tumours that resemble the architecture of healthy tissue.

DISCUSSION
Here, we demonstrate that METTL1, by methylating tRNAs, is vital for regulating cell viability and stress responses.Loss of METTL1- mediated 7-methylguanosine in tRNAs leads to the accumulation of stress-related small noncoding RNAs derived from 5'tRFs, disrupting proteostasis and catabolic cell states.Consequently, METTL1-deficient cells exhibit heightened sensitivity to stress stimuli and display imbalances in redox regulation, increased DNA damage, and senescence.Inhibiting METTL1 expression in tumours impedes their progression and reverts them to a state resembling healthy tissue.Furthermore, METTL1 inhibition renders cancer cells more susceptible to the effects of chemotherapeutic agents, offering potential in combination therapy for enhanced effectiveness.These benefits are particularly relevant in PCa, which ranks as the second most diagnosed cancer among men worldwide, and in patients diagnosed with mCRPC, the 5-year survival rate is 30%.For a long time, Docetaxel in combination with prednisone has been the primary therapeutic option for these patients.However, the heterogeneity of mCRPC necessitates the identification of new targetable molecular pathways to improve treatment outcomes [56,57].Targeting METTL1 presents an attractive molecular approach as it is overexpressed in PCa.METTL1 is also upregulated in other tumour types [21][22][23][24][25][26][27][28][29][40][41][42], which highlights the potential of METTL1 inhibitors as novel therapeutic options in cancer.Moreover, METTL1 expression levels could serve as a valuable biomarker to stratify patients as responders or non-responders to Docetaxel-or rapamycin-based therapies.Further investigations into the relationship between METTL1 expression and response to chemotherapy will be valuable in refining personalised cancer therapeutics.
The development of targeted inhibitors for specific enzymes involved in RNA modifications holds great potential for therapeutic interventions in cancer.While there are currently no METTL1specific inhibitors available, recent breakthroughs in the field of epitranscriptomics and cancer have led to the successful development of inhibitors targeting METTL3, an RNA 6-methyladenosine transferase [58].These inhibitors have shown promising results in preclinical models of leukaemia, demonstrating the therapeutic value of targeting RNA modification enzymes.The successful development of METTL3 inhibitors provides a promising starting point for the design of inhibitors targeting other METTL members, including METTL1.The recent structural characterisation of the METTL1-WDR4 complex and the elucidation of the RNA recognition and methylation mechanism have further enhanced our understanding of METTL1's function [59,60], which provides valuable insights for the development of specific inhibitors.
Recent studies have also revealed that METTL1 inhibition is associated with increased sensitivity to chemotherapy, although the specific functions and molecular mechanisms of tRNA m 7 G methylation remain largely unknown [15, 21-28, 40, 42].In our current research, we have discovered that METTL1 plays a crucial role in regulating the production of 5'tRFs, which trigger a stress response in cancer cells, ultimately leading to enhanced sensitivity to genotoxic agents.These stress-induced 5'tRFs are specifically generated to repress protein translation [14].While the cellular factors governing tRNA cleavage are not yet well understood, recent studies have highlighted the importance of m 5 C, pseudouridine, and adenine methylation as critical determinants of tRNA fate under stress conditions [14,61,62].For instance, NSUN2-mediated methylation of tRNAs regulates the generation of 5'tRFs, which in turn activate stress signalling, induce proteostatic stress, and ultimately decrease the functionality and stress sensitivity of stem and cancer stem cells [18,19].Therefore, our findings, combined with previous reports, indicate that maintaining tRNA integrity and tRNA modifications are essential for cell survival, and disrupting these processes in combination with current chemotherapeutic options could offer more effective strategies for eliminating cancer cells.
METTL1 is highly expressed in various types of cancer, highlighting its significant role in tumorigenesis.However, it is still unclear whether METTL1 is involved in the survival of cancer cells [21][22][23][24][25][26][27][28][40][41][42].Only recent studies have suggested that targeting autophagy could be a promising approach for treating tumours with high METTL1 expression [26].Nevertheless, the precise mechanism by which METTL1 connects the regulation of catabolic pathways and the fitness of cancer cells remains poorly understood.In our study, we uncover that METTL1-mediated tRNA methylation plays a role in the survival and catabolic states of cells by posttranscriptionally regulating the generation of stressassociated 5'tRFs.Interestingly, the abnormal accumulation of these 5'tRFs has been linked to metabolic and neurological disorders, further emphasising their significance [63].
Our findings also demonstrate that METTL1 regulates stress pathways associated with proteostatic stress, autophagy, redox signalling, DNA damage, and senescence [44].These pathways work together to enable cancer cells to adapt to severe stress conditions.Here, we report that METTL1 depletion leads to autophagy inhibition, elevated ROS production, and DNA damage, suggesting that combining METTL1 inhibitors with chemotherapy can improve therapeutic responses in cancer.
Excessive DNA damage can induce both cell death or cell cycle arrest through senescence [64], which has both tumoursuppressive and drug-tolerant persistence characteristics [65,66].These issues can be overcome by combination therapies, since higher doses have been described to induce apoptosis of cells, which can be further eliminated by treatment with senolytic compounds [67].This suggests a potential strategy for METTL1overexpressing tumours, where the use of combination therapies, including METTL1 inhibitors, may synergise with chemotherapy or senolytic therapy to promote more effective elimination of tumour-initiating cells and persistent cancer cells.
In conclusion, our findings unveil a post-transcriptional mechanism for controlling essential functions in cancer cells and uncover novel therapeutic strategies to enhance tumour cell lethality.
Induction was achieved after 3 days of treatment with 0.1-1 µg/ml of doxycycline (Santa Cruz).Silencing or expression efficiencies were tested by RT-qPCR and Western blot.m 7 G methylation analysis Total RNA was extracted using Trizol (Honeywell, 33539), followed by DNAse I Turbo (ThermoFisher Scientific), and tRNAs size-selection using mirVana (ThermoFisher Scientific).tRNAs were de-aminoacylated in 1 mM EDTA, 0.1 M Tris-HCl pH 9.0, for 30 min at 37 °C.tRNAs were used for library preparation or treated with 0.2 M Tris-HCl pH, 0.01 M MgCl 2 , 0.2 M KCl and 10 μg of m 7 GMP (Sigma) for 5 min at 85 °C [68].0.5 M NaBH 4 was added and followed by 30 min ice incubation with aniline (Sigma-Aldrich).T4 PNK (NEB) treatment was followed by library preparation using NEXTFLEX Small RNA-Seq Kit v3 (Bioo Scientific Corp.).All samples were multiplexed and sequenced in HiSeq2500 (Illumina).
Raw sequencing FASTQ files were trimmed with 'cutadapt' retaining reads with a minimum length of 23 nt.Reads were subsequently trimmed by 4 nt on both ends following platform recommendations.Reads were aligned to the GRCh38 (hg38) reference genome using bowtie (bowtiebio.sourceforge.net)with parameters '-m 500 -v 2', allowing a maximum of 500 multiple mappings and two mismatches.tRNA genomic coordinates on hg38 were obtained from the 'GtRNAdb' database (gtrnadb.ucsc.edu).Reads on tRNA coordinates were processed using 'htseq-count' from the Python package 'HTSeq' (htseq.readthedocs.io)with the '-samout' option and counts for individual reads representing tRFs and their multiple sequence alignments on tRNAs were extracted from the resulting sam files using custom PERL scrips.Fragments with less than 10 counts over all samples were removed and a pseudo-count of 1 was added.Count data of tRFs, which were shorter than 0.7 of the full tRNA length, was normalised, and differential abundance of tRFs was evaluated for 3 replicates per condition or treatment using the R package 'DESeq2' (https:// bioconductor.org/packages/release/bioc/html/DESeq2.html).To determine tRNA fragmentation at m 7 G position, we applied the statistical model in 'DESeq2' with 'design ~(condition + treated + condition:treated', where condition represented KO and WT).

Mouse experimental procedures
Mice were maintained at the Animal Research Core Facility at the University of Salamanca, in ventilated filter cages under Specific Pathogen Free conditions with food and water ad libitum.All mouse experiments were performed following the ethical guidelines established by the Biosafety and Bioethics Committee at the University of Salamanca and by the Competent Authority of the Castilla y León Government.Conditional Mettl1 knock out mice were generated as indicated in the supplementary information.

Statistical analysis
GraphPad Prism 8.2 software was used.For in vitro experiments, continue and normally distribution was considered; applying unpaired one-or twotailed Student t-test.For in vivo experiments with ≤10 replicates, parametric Student t-test was applied, while for experiments with >10 replicates unpaired Student t-test was applied for normal distribution and Mann Whitney test for non-normal distribution.Normality was assessed with D'Agostino & Pearson and Shapiro-Wilk tests.For comparison of multiple samples, one-way or two-way ANOVA test was applied.No statistics were applied to determine sample size.In xenograft experiments, mice sacrificed before the end of the experiment were excluded from the analysis.
More methods in supplementary information.

Fig. 1
Fig. 1 METTL1-mediated methylation protects tRNAs from stress-induced cleavage.A Mapping of METTL1-mediated guanosine-7 methylation in tRNAs of PC3-WT and METTL1-KO cells using NaBH 4 /Aniline-induced fragmentation analysis.The start position of statistically significant fragments formed in WT vs. METTL1-KO RNAs after NaBH 4 -treatment is indicated by grey dots.The red arrow indicates METTL1methylated guanosines, the red star represents methylated guanosines of ArgTCT, IleTAT or TyrGTA which contain longer introns and variable loops, and hence position G46 corresponds to later positions that nucleotide 46.B Graphical summary showing tRNA isoacceptors methylated by METTL1 (red), and non-methylated or non-transcribed (black).C, D Detection and quantification of full-length Cys tRNA (upper bands) and 5'tRNA derived fragments (tRFs) using Northern blot analysis in PC3-WT and METTL1-KO cells under unexposed conditions (0 h) or exposed to oxidative stress (NaAsO 2 ) for 2 and 8 h.Two types of 5'tRNA fragments (5'tRFs), long and short, were detected and quantified separately in D. Quantification was further normalised to full-length tRNAs.Detection (E) and quantification (F) of full-length Cys tRNA and 5'tRFs using Northern blot analysis in PC3-WT and METTL1-KO cells under unexposed conditions (0 h) or exposed to NaAsO 2 for 2 h in the presence (+) or absence (-) of 96 μM of angiogenin inhibitor N65828 (ANGi).Long and short 5'tRFs were quantified together in F. Mean ± SEM, n = 2.Total tRNAs were stained with Red safe (C, E).G Protein synthesis rate in PC3-WT and METTL1-KO cells under unexposed conditions (0 h) or after exposure to NaAsO 2 , normalised with cycloheximide (CHX)-treated cells.Mean ± SD, n = 3. H Immunofluorescence of protein aggresomes and CTCF quantification in PC3-WT and METTL1-KO cells (left panel).Scale bar: 50 μm.Mean ± SD, n = 3. Stats: one-tailed Student's t-test (F, G, H), ns: non-significative, *p < 0.05, **p < 0.01.