RESUMO
Despite the discovery of modified nucleic acids nearly 75 years ago, their biological functions are still being elucidated. N6 -methyladenosine (m6 A) is the most abundant modification in eukaryotic messenger RNA (mRNA) and has also been detected in non-coding RNAs, including long non-coding RNA, ribosomal RNA, and small nuclear RNA. In general, m6 A marks can alter RNA secondary structure and initiate unique RNA-protein interactions that can alter splicing, mRNA turnover, and translation, just to name a few. Although m6 A marks in human RNAs have been known to exist since 1974, the structures and functions of methyltransferases responsible for writing m6 A marks have been established only recently. Thus far, there are four confirmed human methyltransferases that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the N6 position of adenosine, producing m6 A: methyltransferase-like protein (METTL) 3/METTL14 complex, METTL16, METTL5, and zinc-finger CCHC-domain-containing protein 4. Though the methyltransferases have unique RNA targets, all human m6 A RNA methyltransferases contain a Rossmann fold with a conserved SAM-binding pocket, suggesting that they utilize a similar catalytic mechanism for methyl transfer. For each of the human m6 A RNA methyltransferases, we present the biological functions and links to human disease, RNA targets, catalytic and kinetic mechanisms, and macromolecular structures. We also discuss m6 A marks in human viruses and parasites, assigning m6 A marks in the transcriptome to specific methyltransferases, small molecules targeting m6 A methyltransferases, and the enzymes responsible for hypermodified m6 A marks and their biological functions in humans. Understanding m6 A methyltransferases is a critical steppingstone toward establishing the m6 A epitranscriptome and more broadly the RNome. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
RESUMO
Recent studies suggest noncoding RNAs interact with genomic DNA, forming RNAâ¢DNA-DNA triple helices, as a mechanism to regulate transcription. One way cells could regulate the formation of these triple helices is through RNA modifications. With over 140 naturally occurring RNA modifications, we hypothesize that some modifications stabilize RNAâ¢DNA-DNA triple helices while others destabilize them. Here, we focus on a pyrimidine-motif triple helix composed of canonical Uâ¢A-T and Câ¢G-C base triples. We employed electrophoretic mobility shift assays and microscale thermophoresis to examine how 11 different RNA modifications at a single position in an RNAâ¢DNA-DNA triple helix affect stability: 5-methylcytidine (m5C), 5-methyluridine (m5U or rT), 3-methyluridine (m3U), pseudouridine (Ψ), 4-thiouridine (s4U), N 6-methyladenosine (m6A), inosine (I), and each nucleobase with 2'-O-methylation (Nm). Compared to the unmodified Uâ¢A-T base triple, some modifications have no significant change in stability (Umâ¢A-T), some have â¼2.5-fold decreases in stability (m5Uâ¢A-T, Ψâ¢A-T, and s4Uâ¢A-T), and some completely disrupt triple helix formation (m3Uâ¢A-T). To identify potential biological examples of RNAâ¢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 â¼1.7-fold with the substitution of m5C at known m5C sites. Therefore, the formation and stability of cellular RNAâ¢DNA-DNA triple helices could be influenced by RNA modifications.
