A single natural RNA modification can destabilize a U•A-T-rich RNA•DNA-DNA triple helix

Abstract

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA·DNA-DNA triple helix. With at least one RNA·DNA-DNA triple helix predicted in the promoter regions of most human genes, RNA·DNA-DNA triple helices may be a common mechanism for regulating transcription. Additionally, cells could employ RNA modifications to regulate the formation of these triple helices. With over 143 naturally occurring RNA modifications, we hypothesize that some modifications stabilize RNA·DNA-DNA triple helices while others destabilize them. Here, we focus on a pyrimidine-motif triple helix composed of canonical U·A-T and C·G-C base triples. We employed electrophoretic mobility shift assays and microscale thermophoresis to examine how eleven different RNA modifications at a single position in an RNA·DNA-DNA triple helix affect stability: 5-methylcytidine (m5C), 5-methyluridine (m5U or rT), 3-methyluridine (m3U), pseudouridine (Ψ), 4-thiouridine (s4U), N6-methyladenosine (m6A), inosine (I), and each nucleobase with 2'-O-methylation (Nm). Compared to the unmodified U·A-T base triple, some modifications have no significant change in stability (Um·A-T), some have ~2.5-fold decreases in stability (m5U·A-T, Ψ·A-T, and s4U·A-T), and some completely disrupt triple helix formation (m3U•A-T). To identify potential biological examples of RNA·DNA-DNA triple helices controlled by an RNA modification, we searched RMVar, a database for RNA modifications mapped at single-nucleotide resolution, for lncRNAs containing an RNA modification within a pyrimidine-rich sequence. Using electrophoretic mobility shift assays, the binding of DNA-DNA to a 22-mer segment of human lncRNA Al157886.1 was destabilized by ~1.7-fold with the substitution of m5C at known m5C sites. Therefore, the formation and stability of cellular RNA·DNA-DNA triple helices could be influenced by RNA modifications.

Global RNA modifications to the MALAT1 triple helix differentially affect thermostability and weaken binding to METTL16

“…For Ψ, UV thermal denaturation assays show that a dA•Ψ-dA inverted motif (where a pyrimidine is located in the central position) is stabilized compared to that of dA•U-dA ( 26 ). In the context of an R•D-D triple helix, a single Ψ•A-T or Ψ•G-C did not significantly impact triple helix stability ( 27 ). Thus, our study provides early insights into how m 6 A, Ψ, and m 1 Ψ affect the stability of a U•A-U-rich triple helix: m 6 A is a destabilizer, Ψ is a stabilizer, and m 1 Ψ stabilizes the Watson–Crick, but not Hoogsteen, face of a major-groove triple helix.…”