N4-Allylcytidine: a new nucleoside analogue for RNA labelling and chemical sequencing.

RNA labelling has become indispensable in studying RNA biology. Nucleoside analogues with a chemical sequencing power represent desirable RNA labelling molecules because precise labelling information at base resolution can be obtained. Here, we report a new nucleoside analogue, N4-allylcytidine (a4C), which is able to tag RNA through both in vitro and in vivo pathways and further specifically reacts with iodine to form 3, N4-cyclized cytidine (cyc-C) in a catalyst-free, fast and complete manner. Full spectroscopic characterization concluded that cyc-C consisted of paired diastereoisomers with opposite chiral carbon centers in the fused 3, N4-five-membered ring. During RNA reverse transcription into complementary DNA, cyc-C induces base misincorporation due to the disruption of canonical hydrogen bonding by the cyclized structure and thus can be accurately identified by sequencing at single base resolution. With the chemical sequencing rationale of a4C, successful applications have been performed including pinpointing N4-methylcytidine methyltransferases' substrate modification sites, metabolically labelling mammalian cellular RNAs, and mapping active cellular RNA polymerase locations with the chromatin run-on RNA sequencing technique. Collectively, our work demonstrates that a4C is a promising molecule for RNA labelling and chemical sequencing and expands the toolkit for studying sophisticated RNA biology.

An RNA Methylation-Sensitive AIEgen-Aptamer Reporting System for Quantitatively Evaluating m6A Methylase and Demethylase Activities.

N6-Methyladenosine (m6A) chemical modification determines the fate of the mammalian cellular mRNA to modulate crucial physiological and pathological processes. Dysregulations of m6A methylase and demethylase have been linked to cancer diseases. Therefore, evaluations of enzyme mutants' activities and related inhibitors for discovery of targeted therapeutic strategies are very necessary. Here, we report an RNA methylation-sensitive fluorescent aptamer reporting assay to measure the catalytic activities of m6A enzymes under various conditions. The rationale is that when an RNA aptamer, named A-Pepper, is methylated at a specific adenosine position to generate m6A-Pepper, the latter displays stronger fluorescence than the former upon binding the ligand, which is an aggregation-induced emission-active luminogen. The fluorescence signal enhancement is linearly proportional to the RNA methylation extent, which is equivalent to the methylase activity. On the contrary, the m6A demethylase activity is measured through calculating the fluorescence signal decrease caused by the switching from m6A-Pepper to A-Pepper. The assay has been successfully applied to quantitatively evaluate the mutation and inhibitor effects on the activities of m6A methylases METTL3/METTL14 and demethylase FTO, and the obtained results are well-consistent with those quantified by the expensive and time-consuming golden standard LC-MS/MS. Our work provides a simple tool capable of detecting m6A enzymes' activities and screening their inhibitors in a rapid, quantitative, cost-effective, and high-throughput manner.

Interactions between host epithelial cells and Acinetobacter baumannii promote the emergence of highly antibiotic resistant and highly mucoid strains.

Acinetobacter baumannii is an important nosocomial pathogen. Upon colonizing a host, A. baumannii are subjected to selective pressure by immune defenses as they adapt to the host environment. However, the mechanism of this pathoadaptation is unknown. Here, we established an in vitro system to evolve A. baumannii driven by the continuous selective pressure exerted by epithelial cells, and we used a combination of experimental evolution, phenotypic characterization and multi-omics analysis to address the underlying mechanism. When continuously exposed to selective pressure by pulmonary epithelial cells, A. baumannii showed ptk mutation-mediated mucoid conversion (reduced adhesion and increased anti-phagocytic ability) by enhancement of capsular exopolysaccharide chain length; rsmG mutation-mediated deficiency of 7-methylguanosine modification in the 524th nucleotide of 16S rRNA, which increased ribosome translation efficiency; and rnaseI mutation-mediated changes in outer membrane permeability and efflux pump expression. Together, these mutations altered susceptibility to a variety of antimicrobial agents, including the novel antibiotic cefiderocol, by regulating siderophore and siderophore-receptor biosynthesis. In conclusion, pulmonary epithelial cells modulate A. baumannii pathoadaptation, implicating the host-microbe interaction in the survival and persistence of A. baumannii.

Cryo-EM structures of human m6A writer complexes.

N6-methyladenosine (m6A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m6A "writer" consists of the catalytic subunit m6A-METTL complex (MAC) and the regulatory subunit m6A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-Å resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA. Furthermore, the 4.4-Å resolution cryo-EM map of the MACOM-MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m6A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m6A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m6A writer complex.

Targeted Manipulation of Cellular RNA m6A Methylation at the Single-Base Level.

Development of tools for precise manipulation of cellular mRNA m6A methylation at the base level is highly required. Here, we report an RNA-guided RNA modification strategy using a fusion protein containing deactivated nuclease Cas13b and m6A methyltransferase METTL14, namely, dCas13b-M14, which is designedly positioned in the cytoplasm. dCas13b-M14 naturally heterodimerizes with endogenous METTL3 to form a catalytic complex to methylate specific cytoplasmic mRNA under a guide RNA (gRNA). We developed assays to screen and validate the guiding specificity of varied gRNAs at single-base resolution. With an optimum combination of dCas13b-M14 and gRNAs inside cells, we have successfully tuned methylation levels of several selected mRNA m6A sites. The off-target effect was evaluated by whole transcriptome m6A sequencing, and a very minor perturbation on the methylome was revealed. Finally, we successfully utilized the editing tool to achieve de novo methylations on five selected mRNA sites. Together, this study paves the way for studying position-dependent roles of m6A methylation in a particular transcript.

New Chromatin Run-On Reaction Enables Global Mapping of Active RNA Polymerase Locations in an Enrichment-free Manner.

The development of a simple and cost-effective method to map the distribution of RNA polymerase II (RNPII) genome-wide at a high resolution is highly beneficial to study cellular transcriptional activity. Here we report a mutation-based and enrichment-free global chromatin run-on sequencing (mGRO-seq) technique to locate active RNPII sites genome-wide at near-base resolution. An adenosine triphosphate (ATP) analog named N6-allyladenosine triphosphate (a6ATP) was designed and could be incorporated into nascent RNAs at RNPII-located positions during a chromatin run-on reaction. By treatment of the run-on RNAs with a mild iodination reaction and subjection of the products to reverse transcription into complementary DNA (cDNA), base mismatch occurs at the original a6A incorporation sites, thus making the RNPII locations detected in the high-throughput cDNA sequencing. The mGRO-seq yields both the map of RNPII sites and the chromatin RNA abundance and holds great promise for the study of single-cell transcriptional activity.

Synthetic modified messenger RNA for therapeutic applications.

Synthetic modified messenger RNA (mRNA) has manifested great potentials for therapeutic applications such as vaccines and gene therapies, with the recent mRNA vaccines for global pandemic COVID-19 (corona virus disease 2019) attracting the tremendous attention. The chemical modifications and delivery vehicles of synthetic mRNAs are the two key factors for their in vivo therapeutic applications. Chemical modifications like nucleoside methylation endow the synthetic mRNAs with high stability and reduced stimulation of innate immunity. The development of scalable production of synthetic mRNA and efficient mRNA formulation and delivery strategies in recent years have remarkably advanced the field. It is worth noticing that we had limited knowledge on the roles of mRNA modifications in the past. However, the last decade has witnessed not only new discoveries of several naturally occurring mRNA modifications but also substantial advances in understanding their roles on regulating gene expression. It is highly necessary to reconsider the therapeutic system made by synthetic modified mRNAs and delivery vectors. In this review, we will mainly discuss the roles of various chemical modifications on synthetic mRNAs, briefly summarize the progresses of mRNA delivery strategies, and highlight some latest mRNA therapeutics applications including infectious disease vaccines, cancer immunotherapy, mRNA-based genetic reprogramming and protein replacement, mRNA-based gene editing. STATEMENT OF SIGNIFICANCE: The development of synthetic mRNA drug holds great promise but lies behind small molecule and protein drugs largely due to the challenging issues regarding its stability, immunogenicity and potency. In the last 15 years, these issues have beensubstantially addressed by synthesizing chemically modified mRNA and developing powerful delivery systems; the mRNA therapeutics has entered an exciting new era begun with the approved mRNA vaccines for the COVID-19 infection disease. Here, we provide recent progresses in understanding the biological roles of various RNA chemical modifications, in developing mRNA delivery systems, and in advancing the emerging mRNA-based therapeutic applications, with the purpose to inspire the community to spawn new ideas for curing diseases.

A Mutation-Based Method for Pinpointing a DNA N6 -Methyladenine Methyltransferase Modification Site at Single Base Resolution.

DNA N6 -methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme-assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl-substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N6 -position of adenine within a specific DNA sequence to form N6 -allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N6 -cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA-containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single-base resolution.

Precise identification of an RNA methyltransferase's substrate modification site.

Here we report a simple and nonradioactive biochemical assay which is capable of accurately determining the substrate methylation sites of human RNA N6-methyladenosine methyltransferases METTL3/METTL14 and METTL16. This method employs enzyme-assisted chemical labelling of a specific base in an RNA substrate with the assistance of an allyl-substituted methyltransferase cofactor, and enables precise identification of the labelling site by a mutation signal from standard nucleic acid sequencing. Our method provides a platform to investigate the enzymatic methylations of long and structurally complex RNA substrates, and facilitates the discovery of new methyltransferases.

A metabolic labeling method detects m6A transcriptome-wide at single base resolution.

Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resolution remains an issue, impeding our understanding of m6A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resolution, called 'm6A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. We pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.

Visualization and quantification of cellular RNA production and degradation using a combined fluorescence and mass spectrometry characterization assay.

Here we report a combined fluorescence and mass spectrometry assay which is capable of stably visualizing and quantifying cellular nucleoside-labeled RNA production and degradation. The fluorescence and mass spectrometry signals from cellular labeled RNAs show a linear correlation. This simple and robust assay benefits the biological community to study RNA metabolism.

VIRMA mediates preferential m6A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation.

N6-methyladenosine (m6A) is enriched in 3'untranslated region (3'UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3'UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3'UTR. Depletions of VIRMA and METTL3 induce 3'UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3'UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3'UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3'UTR and its correlation with alternative polyadenylation.