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
Adenosine, guanosine, cytidine, and uridine can be modified at various positions (red) with a myriad of functional groups (outside circle).
Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 ...are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3′ triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a k cat of 0.07 min–1, and single-turnover assays established a k chem of 0.56 min–1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a k chem of 0.42 min–1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.
Over 140 RNA modifications have been discovered, yet only recently have they been studied in depth due to recent technological advancements. N6‐methyladenosine (m6A) is an abundant RNA modification ...in messenger RNA (mRNA) and long non‐coding RNA that affects various cellular functions such as mRNA stability, phase‐separation of RNAs, and others. Methyltransferase‐like protein 16 (METTL16) is one of four catalytically active m6A RNA methyltransferases in humans. Two well‐known methylation targets of METTL16 are U6 spliceosomal RNA and hairpins in the 3′ untranslated region of MAT2A mRNA. However, METTL16 binds to many other RNAs, including the 3′ triple helix of MALAT1. Using in vitro assays, we have started to investigate the kinetic mechanism and other fundamental properties of METTL16. Thus far, we have determined that METTL16 is a monomer in complex with either U6 snRNA or the MALAT1 triple helix. The METTL16•RNA complex has a dissociation constant (KD) of 18 nM with the U6 snRNA and 31 nM with the MALAT1 triple helix. The apparent dissociation constant for S‐adenosylmethionine (SAM), the methyl donor, with the METTL16•U6 snRNA binary complex is 112 µM. Under in vitro conditions, the cancer‐associated MALAT1 triple helix is not a substrate of METTL16 at position A8290 and other adenosine residues. Preincubation assays suggest that there is an ordered mechanism by which U6 snRNA binds to METTL16 before SAM. Steady‐state assays established a kcat of 0.074 min‐1 and single‐turnover assays established a kchem of 0.56 min‐1. This difference in the rates suggests conformational rearrangements and/or product release may be rate limiting. Ongoing work includes the characterization of METTL16 mutants. Mutations in the METTL16 K‐loop led to a 7‐fold increase of SAM binding to METTL16•U6 snRNA. Future studies will focus on more METTL16 mutants, including critical residues in other structures and those identified in cancer patients, to ascertain how these residues affect the kinetic mechanism of METTL16. This study will enable future research on METTL16 as a therapeutic target.
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