Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA

Mao, Song; Sekula, Bartosz; Ruszkowski, Milosz; Ranganathan, Srivathsan V.; Haruehanroengra, Phensinee; Wu, Ying; Shen, Fusheng; Sheng, Jia

doi:10.1093/nar/gkaa737

Title: Base pairing, structural and functional insights into N⁴-methylcytidine (m⁴C) and N⁴,N⁴-dimethylcytidine (m⁴₂C) modified RNA

Journal Article · Thu Sep 17 00:00:00 EDT 2020 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkaa737· OSTI ID:1682353

Mao, Song ^[1]; Sekula, Bartosz ^[2]; Ruszkowski, Milosz ^[3]; Ranganathan, Srivathsan V. ^[4]; Haruehanroengra, Phensinee ^[1]; Wu, Ying ^[1]; Shen, Fusheng ^[1];

^[1]

State Univ. of New York (SUNY), Albany, NY (United States); State Univ. of New York (SUNY), Albany, NY (United States). The RNA Inst.
National Cancer Inst., Argonne, IL (United States). Macromolecular Crystallography Lab.
National Cancer Inst., Argonne, IL (United States). Macromolecular Crystallography Lab.; Polish Academy of Sciences, Poznan (Poland)
State Univ. of New York (SUNY), Albany, NY (United States). The RNA Inst.

The N⁴-methylation of cytidine (m⁴C and m⁴₂C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m⁴C and m⁴₂C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m⁴C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m⁴₂C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m⁴₂C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N⁴-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Science Foundation (NSF)

Grant/Contract Number:: W-31-109-ENG-38; CHE-1845486; MCB-1715234; NSF-1715234

OSTI ID:: 1682353

Journal Information:: Nucleic Acids Research, Vol. 48, Issue 18; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Title: Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA

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Title: Base pairing, structural and functional insights into N⁴-methylcytidine (m⁴C) and N⁴,N⁴-dimethylcytidine (m⁴₂C) modified RNA