The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as the "fifth" ribonucleotide in 1951. Since then, the ever-increasing number and complexity of modified ribonucleosides have been found in viruses and throughout all three domains of life. Such modifications can be as simple as methylations, hydroxylations, or thiolations, complex as ring closures, glycosylations, acylations, or aminoacylations, or unusual as the incorporation of selenium. While initially found in transfer and ribosomal RNAs, modifications also exist in messenger RNAs and noncoding RNAs. Modifications have profound cellular outcomes at various levels, such as altering RNA structure or being essential for cell survival or organism viability. The aberrant presence or absence of RNA modifications can lead to human disease, ranging from cancer to various metabolic and developmental illnesses such as Hoyeraal-Hreidarsson syndrome, Bowen-Conradi syndrome, or Williams-Beuren syndrome. In this review article, we summarize the characterization of all 143 currently known modified ribonucleosides by describing their taxonomic distributions, the enzymes that generate the modifications, and any implications in cellular processes, RNA structure, and disease. We also highlight areas of active research, such as specific RNAs that contain a particular type of modification as well as methodologies used to identify novel RNA modifications.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA–DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA–DNA triple helices is not well understood beyond the canonical U•A–T and C•G–C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X–Y (where Z = C, U, A, G and X–Y = A–T, G–C, T–A, C–G), in an RNA•DNA–DNA triple helix was determined. The canonical U•A–T and C•G–C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA–DNA triple helix can tolerate up to two consecutive non-canonical A•G–C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA–DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA–DNA and RNA•RNA–RNA triple helices was distinctly different from those in RNA•DNA–DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.

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.

The formation of pyrimidine‐motif RNA●DNA‐DNA (R●D‐D) triple helices (or triplexes), in which ‘●’ and ‘‐’ represent Hoogsteen and Watson‐Crick interactions, has been proposed for over 60 years, but the stability of these structures with RNA modifications has yet to be studied. Eleven RNA modifications were chosen for this study based on their presence in human transcripts and their effects on human health: 5‐methylcytidine (m5C), 5‐methyluridine (m5U), pseudouridine (Ψ), 2ʹ‐O‐methyladenosine (Am), 2ʹ‐O‐methylcytidine (Cm), 2ʹ‐O‐methylguanosine (Gm), 2ʹ‐O‐methyluridine (Um), 3‐methyluridine (m3U), 4‐thiouridine (s4U), inosine (I), and N6‐methyladenosine (m6A). Several of these had been previously found to stabilize or destabilize other nucleic acid duplex and triplex structures. Using both native gel‐shift assays and microscale thermophoresis, the relative stability of a single modified position in a pyrimidine‐motif R●D‐D triple helix was measured at neutral pH. All eleven modifications were found to either have no effect or to destabilize the R●D‐D triple helix, ranging from 2‐fold to complete disruption of binding. For each of the canonical R●D‐D base triples (U●A‐T and C●G‐C), the 2ʹ‐O‐methyl modifications were found to be the least destabilizing, whereas those that directly interfered with the Hoogsteen interactions, such as m3U, were the most destabilizing modifications. As the formation of R●D‐D triple helices in promoter regions of DNA leads to transcriptional enhancement and repression, this study reveals that some RNA modifications could potentially inhibit R●D‐D triplex formation as another level of transcriptional regulation.