Base Pairing and Functional Insights into N³-Methylcytidine (m³C) in RNA

Abstract

N 3-methylcytidine (m3C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m3C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m3C modification significantly disrupts the stability of the Watson–Crick C:G pair. Further m3C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m3C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m3C using reverse transcription in vitro shows that N 3-methylation specifies the C:A pair and induces a G to A change using HIV-1-RT, MMLV-RT, and MutiScribe-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis. Our work provides detailed insights into the thermostability of m3C in RNA and a foundation for developing new molecular tools for mapping m3C in different RNA contexts and exploring the biochemical and biomedical potentials of m3C in the design and development of RNA based therapeutics.

Synthesis and Purification of N3‐Methylcytidine (m3C) Modified RNA Oligonucleotides

“…Interestingly, the RNA duplex containing the m 3 C:G pair displays a melting temperature that is similar to those of other mismatch pairs, i.e., C:A, C:U, and C:C, indicating that this modification decreases base‐pairing discrimination and fidelity. Moreover, the results of molecular dynamics simulations are consistent with our experimental results and provide more detailed structural insights on m 3 C base pairing (Mao et al., 2020).…”

“…This article reports the chemical synthesis of the m 3 C phosphoramidite building block and its incorporation into RNA oligonucleotides via solid‐phase synthesis. Its aims are to synthesize, purify, and biochemically characterize the m 3 C‐modified RNA oligonucleotide as per our recent publication (Mao et al., 2020).…”

METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

“…The precise function of m3C modification in tRNA remains enigmatic. However, biophysical modeling studies suggest that the m3C modification could impact tRNA structure (Mao et al 2021). To investigate a role for m3C in the folding of mt-tRNA-Ser-UGA and mt-tRNA-Thr-UGU, total RNA from WT or METTL8-KO human cells was resolved on non-denaturing native polyacrylamide gels followed by Northern blotting (Woodson and Koculi 2009).…”

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

“…For example, the spectra of methylated cytidines m 4 C, m 5 C and C m contain an ion at m/z 515, whereas this ion is absent in m 3 C (Supplementary Figure S5C). Indeed, it has been recently shown that N3 methylation, which is present on the Watson-Crick face of m 3 C, significantly weakens the base-pairing propensity of cytidine [ 35 ], which suggests that steric hindrance may prevent m 3 C dimer ( m/z 515) formation. Furthermore, similar fingerprint ionization patterns for the distinction of isomers have been characterized before using higher-energy collisional dissociation MS [ 36 ].…”