To monitor m3C formation in tRNA, we used the positive hybridization in the absence of modification (PHA) assay (Fig. 5b)43–46. This Northern blot-based assay relies on differential probe hybridization to tRNA caused by the presence or absence of m3C, which impairs base-pairing47–49. Thus, a decrease in m3C modification leads to an increase in PHA probe signal that can be normalized against the probe signal from a different region of the same tRNA as an internal control. For tRNA-Arg-CCU and UCU, we observed a considerable increase in PHA probe signal in the human D3-KO cell line compared to WT, indicating the loss of m3C modification in these particular tRNAs (Fig. 5c). In contrast, no increase in PHA signal was detected for either tRNA-Ser-UGA or tRNA-Thr-AGU in the D3-KO cell line, consistent with the substrate specificity of DALRD3 shown above. We also note that the steady-state levels of all tested tRNAs remained similar between the WT and D3-KO cell lines, including tRNA-Arg-CCU and UCU.
