Structural impact of 3-methylcytosine modification on the anticodon stem of a neuronally-enriched arginine tRNA

Abstract

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 vs 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 suggests 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.

Keywords: 3-methylcytosine; NMR; RNA modification; RNA structure; anticodon stem-loop; m3C; optical melting; tRNA; tRNA-Arg-UCU.

Figure 1.

A: Sequence of neuronal tRNA-Arg-UCU-4-1 isodecoder displaying the anticodon stem loop in black. B: Anticodon stem loops ASLnomod and ASLm3C with their sequence and numbering scheme. C: Structure of the methylated m3C base with the numbering scheme used for referring to the m3C base. D: Imino proton spectra of ASLnomod and ASLm3C as a function of temperature. The peak labeled m3C32 results from the imine protons at position 4 within the m3C base.

Figure 2.

The change in chemical shift is shown on the Y-axis and the residue number and atom corresponding to that particular chemical shift are shown on the X-axis. Bars are colored according to whether they are part of the stem (white), terminal mismatch (red), or hairpin loop (green).

Figure 3.

A: 75 ms NOESY spectrum of ASL_nomod displaying the unusually strong A37H1’ – A38 H8 NOE (blue). For comparison, other n H1’ – n+1 H8 NOEs (black) are shown to exemplify the average volume of similar resonances. B: Short A37 H1’ – A38 distance results in tilt between A37 and A38 residues. C: Structure of adenine bases in syn and anti conformations are shown along with the corresponding percentage of syn vs anti for ASL_nomod and ASL_m3C.

Figure 4.

A: The two most prevalent sugar conformations in RNA are C3’ endo (black) and C2’ endo (blue). NMR indicates a preference for C2’ endo when significant splitting is seen between the H1’ and H2’ resonances. B: The sugar conformation preference of the residues within ASLnomod and ASLm3C is shown. Residues preferring C2’ endo are shown in blue whereas residues preferring C3’ endo are shown in black.

Figure 5.

Conformation of C32 – A38 pairing before modification (A) and after m3C modification (B) remains the same despite modification. C: NOEs from the m3C methyl group. NOEs shown in green are only visible at long mixing time (400 ms) whereas NOEs shown in orange are visible at short mixing time.

Figure 6.

Shown are long range (∣I – j∣ ≥ 2) NOE connections observed within the loops for ASL_nomod (A, blue) and ASL_m3C (B, red). Shown NOEs include those from both short mixing time (75 ms) and long mixing time (400 ms). Overlaid NMR conformers for ASL_nomod (C) and ASL_m3C (D) demonstrate increased variability within the loop of ASL_m3C due to a lack of long-range NOEs for ASL_m3C.

Figure 7.

A: Lowest energy NMR conformer of ASL_nomod. B: Lowest energy NMR conformer of ASL_m3C C: Structures of ASL_nomod predicted by Alphafold3 and trRosettaRNA. Shown below the predicted structure is the all-atom RMSD and loop RMSD using the lowest energy NMR conformer as a reference. C: Lowest energy ASL_m3C structure modeled with NMR-derived restraints. The corresponding RMSD values for ASL_m3C used the lowest energy ASL_nomod conformer as a reference structure and excluded residue 32 due to the presence of the methyl group.