Significance: RNA triple helices were deduced to form in vitro almost 60 years ago, yet only three examples from eukaryotic cellular RNAs have been structurally validated. The longest triple helix, the MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) ENE+A (element for nuclear expression with a downstream A-rich tract), presents an opportunity to investigate the biological roles of these enigmatic structures. We have discovered that the MALAT1 triple helix is specifically recognized and bound by METTL16 (methyltransferase-like protein 16). There are two important implications: ( i ) the MALAT1 triple helix is a bona fide structure in the cellular environment and ( ii ) there may exist an undiscovered class of triple-stranded RNA binding proteins. METTL16’s antiproliferative role in Caenorhabditis elegans suggests that the METTL16–MALAT1 complex may contribute to the oncogenic activity of MALAT1.

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.This article is categorized under: RNA Processing > RNA Editing and Modification

N⁶-methyladenosine (m⁶A) is an abundant modification in messenger RNA and noncoding RNAs that affects RNA metabolism. Methyltransferase-like protein 16 (METTL16) is a recently confirmed m⁶A RNA methyltransferase that methylates U6 spliceosomal RNA and interacts with the 3′-terminal RNA triple helix of MALAT1 (metastasis-associated lung adenocarcinoma transcript 1). Here, we present two X-ray crystal structures of the N-terminal methyltransferase domain (residues 1–291) of human METTL16 (METTL16_291): an apo structure at 1.9 Å resolution and a post-catalyticS-adenosylhomocysteine-bound complex at 2.1 Å resolution. The structures revealed a highly conserved Rossmann fold that is characteristic of Class IS-adenosylmethionine-dependent methyltransferases and a large, positively charged groove. This groove likely represents the RNA-binding site and it includes structural elements unique to METTL16. In-depth analysis of the active site led to a model of the methyl transfer reaction catalyzed by METTL16. In contrast to the major m⁶A methyltransferase heterodimer METTL3/METTL14, full-length METTL16 forms a homodimer and METTL16_291 exists as a monomer based on size-exclusion chromatography. A native gel-shift assay shows that METTL16 binds to the MALAT1 RNA triple helix, but monomeric METTL16_291 does not. Our results provide insights into the molecular structure of METTL16, which is distinct from METTL3/METTL14.

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an abundant nuclear-localized long noncoding RNA (lncRNA) that has significant roles in cancer. While the interacting partners and evolutionary sequence conservation of MALAT1 have been examined, much of the structure of MALAT1 is unknown. Here, we propose a hypothetical secondary structural model for 8425 nucleotides of human MALAT1 using three experimental datasets that probed RNA structures in vitro and in various human cell lines. Our model indicates that approximately half of human MALAT1 is structured, forming 194 helices, 13 pseudoknots, five structured tetraloops, nine structured internal loops, and 13 intramolecular long-range interactions that give rise to several multiway junctions. Evolutionary conservation and covariation analyses support 153 of 194 helices in 51 mammalian MALAT1 homologs and 42 of 194 helices in 53 vertebrate MALAT1 homologs, thereby identifying an evolutionarily conserved core that likely has important functional roles in mammals and vertebrates. Data mining revealed that RNA modifications, somatic cancer-associated mutations, and single-nucleotide polymorphisms may induce structural rearrangements that sequester or expose binding sites for several cancer-associated microRNAs. Our findings reveal new mechanistic leads into the roles of MALAT1 by identifying several intriguing structure–function relationships in which the dynamic structure of MALAT1 underlies its biological functions.

It has been nearly 63 years since the first characterization of an RNA triple helix in vitro by Gary Felsenfeld, David Davies, and Alexander Rich. An RNA triple helix consists of three strands: A Watson–Crick RNA double helix whose major‐groove establishes hydrogen bonds with the so‐called “third strand”. In the past 15 years, it has been recognized that these major‐groove RNA triple helices, like single‐stranded and double‐stranded RNA, also mediate prominent biological roles inside cells. Thus far, these triple helices are known to mediate catalysis during telomere synthesis and RNA splicing, bind to ligands and ions so that metabolite‐sensing riboswitches can regulate gene expression, and provide a clever strategy to protect the 3′ end of RNA from degradation. Because RNA triple helices play important roles in biology, there is a renewed interest in better understanding the fundamental properties of RNA triple helices and developing methods for their high‐throughput discovery. This review provides an overview of the fundamental biochemical and structural properties of major‐groove RNA triple helices, summarizes the structure and function of naturally occurring RNA triple helices, and describes prospective strategies to isolate RNA triple helices as a means to establish the “triplexome”.This article is categorized under: RNA Structure and Dynamics > RNA Structure and Dynamics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems