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.