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N 1 A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m 1 A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m 1 A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m 1 A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m 1 A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m 1 A. In this study, we introduce a reduction-based m 1 A sequencing (red-m 1 A-seq). We report that NaBH 4 reduction of m 1 A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m 1 A to m 6 A to provide good controls, allowing the detection of m 1 A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m 1 A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m 1 A sites, but also new m 1 A sites in mt-tRNA Asn-GTT and 5.8S rRNA., (© 2024 Pajdzik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Posttranscriptional RNA modifications occur in almost all types of RNA in all life forms. As an abundant RNA modification in mammals, pseudouridine (Ψ) regulates diverse biological functions of different RNA species such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), etc. However, the functional investigation of mRNA pseudouridine (Ψ) has been hampered by the lack of a quantitative method that can efficiently map Ψ transcriptome-wide. We developed bisulfite-induced deletion sequencing (BID-seq), with the optimized bisulfite-based chemical reaction to convert pseudouridine selectively and completely into Ψ-BS adduct without cytosine deamination. The Ψ-BS adduct can be further read out as deletion signatures during reverse transcription. The deletion ratios induced by Ψ sites were used for estimating the modification stoichiometry at each modified site. BID-seq starts with 10-20 ng polyA + RNA and detects thousands of mRNA Ψ sites with stoichiometry information in cell lines and tissues. We uncovered consensus motifs for Ψ in mammalian mRNA and assigned specific 'writer' proteins to individual Ψ deposition. BID-seq also confirmed the presence of Ψ within stop codons of mammalian mRNA. BID-seq set the stage for future investigations of Ψ functions in diverse biological processes., (Copyright © 2023. Published by Elsevier Inc.)

Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m 7 G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m 7 G-quant-seq, a quantitative method that accurately detects internal m 7 G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m 7 G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m 7 G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m 7 G methylation level. We detected internal m 7 G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m 7 G methylation stoichiometry. m 7 G-quant-seq could be applied to monitor the tRNA m 7 G methylation level change in diverse biological processes.

N 6 A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m 6 A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m 6 A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m 6 A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0 to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time- and cost-effective approach for absolute quantification of the m 6 A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.6 A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications., (© 2020 Bujnowska et al.)

RNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N 6 -allyladenosine (a 6 A) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. a 6 A behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells. We also show that human RNA N 6 -methyladenosine (m 6 A) methyltransferases METTL3/METTL14 can work with a synthetic cofactor, namely allyl-SAM (S-adenosyl methionine with methyl replaced by allyl) in order to site-specifically install an allyl group to the N 6 -position of A within specific sequence to generate a 6 A-labeled RNAs. The iodination of N 6 -allyl group of a 6 A under mild buffer conditions spontaneously induces the formation of N 1 ,N 6 -cyclized adenosine and creates mutations at its opposite site during complementary DNA synthesis of reverse transcription. The existing m 6 A in RNA is inert to methyltransferase-assisted allyl labeling, which offers a chance to differentiate m 6 A from A at individual RNA sites. Our work demonstrates a new method for RNA labeling, which could find applications in developing sequencing methods for nascent RNAs and RNA modifications.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and affects almost every stage of the mRNA life cycle. The YTH-domain proteins can specifically recognize m6A modification to control mRNA maturation, translation and decay. m6A can also alter RNA structures to affect RNA-protein interactions in cells. Here, we show that m6A increases the accessibility of its surrounding RNA sequence to bind heterogeneous nuclear ribonucleoprotein G (HNRNPG). Furthermore, HNRNPG binds m6A-methylated RNAs through its C-terminal low-complexity region, which self-assembles into large particles in vitro. The Arg-Gly-Gly repeats within the low-complexity region are required for binding to the RNA motif exposed by m6A methylation. We identified 13,191 m6A sites in the transcriptome that regulate RNA-HNRNPG interaction and thereby alter the expression and alternative splicing pattern of target mRNAs. Low-complexity regions are pervasive among mRNA binding proteins. Our results show that m6A-dependent RNA structural alterations can promote direct binding of m6A-modified RNAs to low-complexity regions in RNA binding proteins., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N(6) -methyladenosine (m(6) A) is an abundant internal modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism. However, methods to precisely locate the m(6) A modification remain limited. We present here a photo-crosslinking-assisted m(6) A sequencing strategy (PA-m(6) A-seq) to more accurately define sites with m(6) A modification. Using this strategy, we obtained a high-resolution map of m(6) A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m(6) A sites at base resolution. Our results provide insight into the relationship between the methylation regions and the binding sites of RNA-binding proteins., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

N(6)-methyladenosine (m(6)A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m(6)A RNA methylation. Together with METTL3, the only previously known m(6)A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m(6)A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the (18)O KIEs on the 2'O nucleophile, 5'O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5'O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5'O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2'-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.

N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.

The 2'-hydroxyl groups within RNA contribute in essential ways to RNA structure and function. Previously, we designed an atomic mutation cycle (AMC) that uses ribonucleoside analogues bearing different C-2'-substituents, including -OCH(3), -NH(2), -NHMe, and -NMe(2), to identify hydroxyl groups within RNA that donate functionally significant hydrogen bonds. To enable AMC analysis of the nucleophilic guanosine cofactor in the Tetrahymena ribozyme reaction and at other guanosines whose 2'-hydroxyl groups impart critical functional contributions, we describe here the syntheses of 2'-methylamino-2'-deoxyguanosine (G(NHMe)) and 2'-N,N-dimethylamino-2'-deoxyguanosine (G(NMe(2))) and their corresponding phosphoramidites. The key step in obtaining the nucleosides involved S(N)2 displacement of 2'-β-triflate from an appropriate guanosine derivative by methylamine or dimethylamine. We readily obtained the G(NMe(2)) phosphoramidite and incorporated it into RNA. However, the G(NHMe) phosphoramidite posed a significantly greater challenge due to lack of a suitable -2'-NHMe protecting group. After testing several strategies, we established that allyloxycarbonyl (Alloc) provided suitable protection for 2'-N-methylamino group during the phosphoramidite synthesis and the subsequent RNA synthesis. This work enables AMC analysis of guanosine's 2'-hydroxyl group within RNA.

To better understand the interactions between catalysts and transition states during RNA strand cleavage, primary (18)O kinetic isotope effects (KIEs) and solvent D(2)O isotope effects were measured to probe the mechanism of base-catalyzed 2'-O-transphosphorylation of the RNA dinucleotide 5'-UpG-3'. The observed (18)O KIEs for the nucleophilic 2'-O and in the 5'-O leaving group at pH 14 are both large relative to reactions of phosphodiesters with good leaving groups, indicating that the reaction catalyzed by hydroxide has a transition state (TS) with advanced phosphorus-oxygen bond fission to the leaving group ((18)k(LG) = 1.034 +/- 0.004) and phosphorus-nucleophile bond formation ((18)k(NUC) = 0.984 +/- 0.004). A breakpoint in the pH dependence of the 2'-O-transphosphorylation rate to a pH independent phase above pH 13 has been attributed to the pK(a) of the 2'-OH nucleophile. A smaller nucleophile KIE is observed at pH 12 ((18)k(NUC) = 0.995 +/- 0.004) that is interpreted as the combined effect of the equilibrium isotope effect (ca. 1.02) on deprotonation of the 2'-hydroxyl nucleophile and the intrinsic KIE on the nucleophilic addition step (ca. 0.981). An alternative mechanism in which the hydroxide ion acts as a general base is considered unlikely given the lack of a solvent deuterium isotope effect above the breakpoint in the pH versus rate profile. These results represent the first direct analysis of the transition state for RNA strand cleavage. The primary (18)O KIE results and the lack of a kinetic solvent deuterium isotope effect together provide strong evidence for a late transition state and 2'-O nucleophile activation by specific base catalysis.

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.

5‐Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt‐tRNAMet) and cytosolic leucine tRNA (ct‐tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has also been detected in yeast mRNA and human tRNA, but its transcriptome‐wide distribution in mammals has not been studied. Here we report f5C‐seq, a quantitative sequencing method to map f5C transcriptome‐wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pic‐borane, and DHU can be exclusively read as T during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C‐to‐T mutation signature. We validated f5C‐seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1‐depleted cells. By applying f5C‐seq to chromatin‐associated RNA (caRNA), we identified several highly modified f5C sites in HeLa and mouse embryonic stem cells (mESC). [ABSTRACT FROM AUTHOR]

We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m6A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m6A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m6A in nuclear RNA is a major physiological substrate of FTO. [ABSTRACT FROM AUTHOR]

A correction to the article " N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO" that was published in the October 16, 2011 issue is presented.

Soil microbial communities play a vital role in mediating nutrient turnover, thus enhancing growth and development of plants. Understanding the dynamics of microorganisms in soils can provide insight into the influence of fertilization practices on improving soil fertility and plant growth in agricultural ecosystems. In this study, we compared the abundances and compositions of total (DNA-based, 16S rRNA gene) and active (RNA-based, 16S rRNA) bacterial communities at a 30-year experimental site in different inorganic fertilization treatments with different key elements (nitrogen, phosphorus, and potassium). The inorganic fertilizer amendments did not affect the abundance of total bacteria but significantly affected the abundance of active bacteria due to changes in microbial biomass carbon and NH 4 +-N contents. Cyanobacteria and Proteobacteria , especially for some dominant orders (e.g. Nostocales , Pseudanabaenales and Nitrosomonadales) were the dominant phyla in the active microbial community and differed proportionally in nitrogen and phosphorus fertilized soil. Soil N speciation (e.g. total N, NH 4 +-N and NO 3 −-N) were the main determinants controlling the Cyanobacteria and Proteobacteria communities. Our results indicated that the unbalanced fertilization could reduce the abundance of active bacteria and significantly changed the dominant phyla compared with balanced fertilization. These findings provided an insight of composition and ratio of nutrient elements including nitrogen, phosphorus and potassium for managing future fertilization regimes in agricultural ecosystem. • The chemical fertilizers obviously affected active bacteria but not total bacteria. • Cyanobacteria and Proteobacteria were dominant in the active microbial community. • N availability was the main driver structuring Cyanobacteria and Proteobacteria. • Active bacteria was more sensitive than total bacteria to environmental change. [ABSTRACT FROM AUTHOR]