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Link to original content: https://pubmed.ncbi.nlm.nih.gov/27478929
m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs - PubMed Skip to main page content
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. 2016 Sep;23(9):803-10.
doi: 10.1038/nsmb.3270. Epub 2016 Aug 1.

m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs

Huiqing Zhou  1 , Isaac J Kimsey  1 , Evgenia N Nikolova  2 , Bharathwaj Sathyamoorthy  1 , Gianmarc Grazioli  3 , James McSally  3 , Tianyu Bai  1 , Christoph H Wunderlich  4 , Christoph Kreutz  5   6 , Ioan Andricioaei  3 , Hashim M Al-Hashimi  1   7
Affiliations

m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs

Huiqing Zhou et al. Nat Struct Mol Biol. 2016 Sep.

Abstract

The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

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Figures

Figure 1
Figure 1
Absence of detectable WC⇄HG exchange in A-RNA by NMR relaxation dispersion. (a) Comparison of A-form RNA (violet) and B-form (blue) DNA double helices. (b) WC and HG bps in dynamic equilibrium in B-DNA. Sites used for RD measurements are highlighted in orange. (c) A6-DNA and hp-A6-RNA duplexes with bps targeted in RD measurements highlighted. (d) Off-resonance RD profiles showing R2+Rex as a function of spin lock offset (Ω 2π−1Hz, where Ω = Ωobs – ωRF) and power (ωSL 2π−1Hz, in insets). Error bars represent experimental uncertainty (one s.d.) estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). Solid line represents a fit to two-state exchange.
Figure 2
Figure 2
Lack of detectable exchange across diverse RNA sequence and structural contexts. (a) Secondary structures with bps showing no detectable RD highlighted in red. (b) Off-resonance RD profiles for the highlighted bps with error bars representing experimental uncertainty (one s.d.) estimated from mono-exponential fitting using a Monte-Carlo based method (Methods).
Figure 3
Figure 3
m1A and m1G do not form HG bps and disrupt A-RNA structure. (a) N1-methylated purines trap HG bps in B-DNA. NMR chemical shift probes of HG bps are in orange and of purine methylation state in cyan. Arrows indicate characteristic HG NOE cross-peaks. (b) Duplexes containing m1A or m1G (turquoise circles). syn or anti purines deduced by NMR are shown as open and filled letters, respectively. HG and partially melted bps as deduced by NMR are indicated using open and dashed lines, respectively. Residues showing significant chemical shift perturbations or line-broadening due to m1A or m1G are colored orange and grey, respectively. (c) m1A or m1G induced purine-C1′ chemical shift perturbations (Δω = ωmodified – ωunmodified) in A-RNA (violet) and B-DNA (blue). Shown for comparison are Δω = ωHG – ωWC measured for transient dA–dT HG bps by RD (“RD”) in unmodified DNA duplexes (error bars showing one s.d.) and computed for adenine residues using DFT (Methods). (d) NOESY H1′–H8 cross-peaks showing syn purine bases in B-DNA but not A-RNA. Shown for reference is the cytosine base H5–H6 NOE with inter-atomic distance ≈2.5 Å. (e) 1D 1H spectra showing the imino/amino resonances expected for HG type H-bonds in A6-DNAm1A and A6-DNAm1G but not in methylated RNA at 5°C and 15°C, respectively. (f) Example showing m1G induced loss of a WC imino resonance (highlighted in a circle) in A6-RNA but not A6-DNA in 2D NMR spectra. (g) Example downfield shifted carbon chemical shifts induced by m1A. (h) Free energy (ΔΔG) and enthalpy (ΔΔH) destabilization due to m1A and m1G in DNA (blue) and RNA (violet) duplexes measured by UV melting experiments with error bars, one s.d. (n = 3 independent measurements) (Methods and Supplementary Table 2).
Figure 4
Figure 4
Source of HG instability in A-RNA. (a) Comparison of RD profiles measured in A6-DNArA, A6-DNArG, and A6-DNA. Error bars correspond to one s.d. estimated from mono-exponential fitting using a Monte-Carlo based method (Methods). Note that the larger R2 value in A6-DNArA A16-C8 as compared to A6-DNA likely reflects decreased flexibility in rA16. Exchange parameters are shown in Supplementary Fig. 4b. (b) Inter-atomic distances (in Å) with unfavorable steric contacts in pink when rotating the purine base 180° around the glycoside bond in WC bps derived from idealized B-DNA and A-RNA duplexes (Methods) to adopt a syn conformation. Shown below are corresponding distance distributions in WC bps derived from X-ray structures of A-RNA (total 146) and B-DNA (total 159) duplexes before (solid line) and following (dashed line) 180° rotation of the purine base. The inter-atomic cut-off distance (grey line) was defined based on the van der Waals radii. (c) Relative interaction energy versus χ-angle from biased MD trajectories of A6-DNA (blue) and hp-A6-RNA (violet). (d) Simulation time (ns) versus the global RMSD (Methods) for single A6-DNAm1A and hp-A6-RNAm1A trajectories depicting the destabilization of the RNA strand within the time of the simulation.
Figure 5
Figure 5
Different propensities to form HG bps in B-DNA and A-RNA enable contrasting roles at the genome and transcriptome level. (a) In DNA, m1dA or m1dG damage is absorbed as HG bps that can be recognized by repair enzymes (in red). Had B-DNA lacked the ability to form HG bps, damage could result in duplex melting and genomic instability. In RNA, post-transcriptional modifications resulting in m1rA and m1rG block both WC and HG pairing, melting or modulating RNA secondary structure to favor functional states or effect epigenetic regulation. Had A-RNA had the ability to form HG, the m1rA and m1rG would form HG bps and potentially fail to more significantly alter RNA structure and function. (b) Highly conserved m1rA9 in human mitochondrial tRNALys blocks rA–rU WC base pairing and stabilizes native tRNA structure in which m1rA9 is in a single strand58. The m1rA9 modification would not stabilize native tRNA structure if it were simply absorbed as a HG bp. (c) Highly conserved m1rG37 next to the anti-codon loop blocks base pairing between m1rG37 and the first rC in the codon and prevents +1 frameshifting in tRNAPro, which could occur if m1rG37 formed stable HG bp with rC. (d) Proposed mechanism for m1rA enhanced translation through destabilization of secondary structure in the 5′ UTR of mRNA.
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