Structural Impact of 3-methylcytosine Modification on the Anticodon Stem-loop of a Neuronally-enriched Arginine tRNA

All tRNAs undergo a series of chemical modifications to fold and function correctly. In mammals, the C32 nucleotide in the anticodon loop of tRNA-Arg-CCU and UCU is methylated to form 3-methylcytosine (m3C). Deficiency of m3C in arginine tRNAs has been linked to human neurodevelopmental disorders, indicating a critical biological role for m3C modification. However, the structural repercussions of m3C modification are not well understood. Here, we examine the structural effects of m3C32 modification on the anticodon stem loop (ASL) of human tRNA-Arg-UCU-4-1, a unique tRNA with enriched expression in the central nervous system. Optical melting experiments demonstrate that m3C modification can locally disrupt nearby base pairing within the ASL while simultaneously stabilizing the ASL electrostatically, resulting in little net change thermodynamically. The isoenergetic nature of the C32-A38 pair versus the m3C32-A38 pair may help discriminate against structures not adopting canonical C32-A38 pairings, as most other m3C pairings are unfavorable. Furthermore, multidimensional NMR reveals that after m3C modification there are changes in hairpin loop structure and dynamics, the structure of A37, and the neighboring A31-U39 base pair. However, these structural changes after modification are made while maintaining the shape of the C32-A38 pairing, which is essential for efficient tRNA function in translation. These findings suggest that m3C32 modification could alter interactions of tRNA-Arg isodecoders with one or more binding partners while simultaneously maintaining the tRNA's ability to function in translation.