Fig. 4

The lack of m⁷G methylation in tRNAs leads to 5'tRNA fragment accumulation. A Heatmap showing no differences in the levels of mature tRNAs (log₂ normalised reads; RPKM) measured using tRNA-seq data. Rows represent individual tRNA isoacceptors and columns represent independent replicates. tRNA isoacceptor expression levels are row-scaled (each row is normalised to their mean expression and standard variation). B The top pie chart represents the percentage of all abundant tRNA fragments identified in PC3 METTL1 KO cells compared to that in WT cells. The bottom pie chart represents the percentage of differentially express tRNA fragments: fragments with log₂ fold change (FC) > 2 and p value < 0.05 in PC3 METTL1 KO cells versus WT cells. The pie charts show that while 5'tRNA fragments are very abundant in KO cells, 5'TOGs are significantly overexpressed in KO cells compared to WT cells. 3'tRFS: 3' tRNA fragments; Int-tRFs: internal tRFs; 5'tRNA: 5'tRNA fragments > 18 and < 35 nucleotides; 5'-halves: 5'tRNA fragments > 35 nucleotides; 5'TOGs: 5'tRNA fragments > 18 and < 35 nucleotides, with 5′ terminal oligoguanine. C The boxplot shows the log₂ fold change (FC) of all fragments (not just the differentially expressed tRNA fragments) in PC3 METTL1 KO versus WT cells. D Size and abundance (density) of 5'TOGs Cys-derived (5G) or Ala-derived (4G) fragments differentially expressed (log₂ FC > 1.5, > 18 nt, and p < 0.05) in PC3 METTL1 KO cells versus WT cells. E Summary of 5'TOGs formed in PC3 METTL1 KO cells. F Increased tRNA fragmentation was observed by northern blot detection of Cys-derived 5’TOGs in PC3 METTL1 KO vs. WT cells (2 technical replicates of 2 biological replicates are represented) (left panel). The boxplot of the right shows quantification of densities of Cys-derived 5'TOGs formed versus full-length tRNAs. G, H Cys-derived 5'TOG fragments are induced by stress. Northern blot detection of Cys-derived 5'TOG in PC3 and DU145 METTL1 KO and WT cells unexposed (0 h), or after 2 and 8 h of oxidative stress exposure. The boxplots of the right show the fraction of Cys-derived 5'tRFs formed normalised to the fraction of full length tRNAs. Values are from two biological replicates, shown in this figure and in Supplementary Fig. S4G and S4J. The loading control of tRNA is shown in the bottom panel as red-safe staining (F–H). Bands corresponding to full length tRNAs are indicated with stars and 5'tRNA fragments are indicated with arrows. Statistical tests: one-tailed Student’s t-test. **p < 0.01 (F–H)