The co-purification of DALRD3 with METTL2A and 2B along with the distinct tRNA-binding specificity of DALRD3 suggests that DALRD3 plays a role in targeting METTL2A/B to specific arginine tRNAs for m3C modification. To test this hypothesis, we transiently expressed Strep-METTL2A or 2B with or without co-expression of FLAG-tagged DALRD3 in 293T cells. Immunoblot analysis of the input extracts confirmed the expression of Strep-METTL2A/B either alone or with FLAG-DALRD3 (Fig. 3a, lanes 2, 3, 5, and 6). Due to the transfection procedure, METTL2A and 2B exhibited reduced levels of expression when co-expressed with DALRD3 compared to METTL2A or 2B expressed alone. Using this approach, we detected the co-purification of FLAG-DALRD3 with Strep-METTL2A or 2B, further corroborating our finding that METTL2A/B interacts with DALRD3 (Fig. 3a, lanes 11 and 12). We also find that the interaction between DALRD3 and METTL2A/B persists after RNase treatment, suggesting that METTL2 interaction with DALRD3 is not bridged through RNA (Supplementary Fig. 2).
