m6A-label-seq: A metabolic labeling protocol to detect transcriptome-wide mRNA N6-methyladenosine (m6A) at base resolution

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N 6 -Methyladenosine (m 6 A) chemical modification determines the fate of the mammalian cellular mRNA to modulate crucial physiological and pathological processes. Dysregulations of m 6 A 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 m 6 A enzymes under various conditions. The rationale is that when an RNA aptamer, named A-Pepper, is methylated at a specific adenosine position to generate m 6 A-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 m 6 A demethylase activity is measured through calculating the fluorescence signal decrease caused by the switching from m 6 A-Pepper to A-Pepper. The assay has been successfully applied to quantitatively evaluate the mutation and inhibitor effects on the activities of m 6 A 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 m 6 A enzymes' activities and screening their inhibitors in a rapid, quantitative, cost-effective, and high-throughput manner.

Section: Results and Discussionmentioning

confidence: 99%

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

Ying,

Huang,

et al. 2023

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“…The larger the m 6 A stoichiometry is, the lower is the amount of MazF-cut RNA products. Based on our single-base resolution m 6 A database obtained from m 6 A-label-seq., ,, we selected several mRNA m 6 A sites for the editing experiment, such as SPEN -3525, SPEN -6281, YTHDF2 -1551, YTHDF3 -1884, GAPDH -782, and HSPA1A -771 (Figure S3). All of these sites showed increased m 6 A levels with varied extents from 1.2- to 2.4-fold in the presence of their respective (−1)-gRNAs (Figures B,C and S3A).…”

Section: Resultsmentioning

confidence: 99%

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

Gao

Cao

et al. 2022

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Development of tools for precise manipulation of cellular mRNA m 6 A 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 m 6 A 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 m 6 A sites. The off-target effect was evaluated by whole transcriptome m 6 A 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 m 6 A methylation in a particular transcript.

“…First, we prepared a 300 nt a 6 A-RNA probe using synthetic a 6 ATP instead of ATP by an in vitro transcription method. The a 6 A in the RNA was stoichiometrically transformed into cyc-A under mild iodination reaction conditions. − HIV reverse transcriptase has been shown to misincorporate a base at the site opposite of cyc-A in RNA. − Here we designed primers for a probe segment containing two a 6 A sites spaced with 15 nt and performed an RT-induced mutation assay. Both the treated cyc-A-RNA probe and the untreated a 6 A-RNA probe were subjected to RT into cDNA followed by polymerase chain reaction (PCR) amplification and TA-clone-based Sanger sequencing.…”

Section: Resultsmentioning

confidence: 99%

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

Gao

Shu

et al. 2022

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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 (mGROseq) technique to locate active RNPII sites genome-wide at nearbase resolution. An adenosine triphosphate (ATP) analog named N 6 -allyladenosine triphosphate (a 6 ATP) 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 a 6 A 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.