Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution

Abstract

SUMMARY Epigenetic modifications control the stability and translation of mRNA molecules. Here, we present a microscopy-based platform for quantifying modified RNA molecules and for relating the modification patterns to single-cell phenotypes. We directly capture mRNAs from cell lysates on oligo-dT-coated coverslips, then visually detect and sequence individual m 6 A-immunolabled transcripts without amplification. Integration of a nanoscale device enabled us to isolate single cells on the platform, and thereby relate single-cell m 6 A modification states to gene expression signatures and cell surface markers. Application of the platform to MUTZ3 leukemia cells revealed a marked reduction in cellular m 6 A levels as CD34 + leukemic progenitors differentiate to CD14 + myeloid cells. We then coupled single-molecule m 6 A detection with fluorescence in situ hybridization (FISH) to relate mRNA and m 6 A levels of individual genes to single-cell phenotypes. This single-cell multi-modal assay suite can empower investigations of RNA modifications in rare populations and single cells.

Unraveling the RNA Tapestry: A Symphony of Innovations in m6A Research Technology

“…In the same year, another method was reported to enable spatially mapping of m 6 A at single-molecule and single-cell level using microscopy-based platforms (Figure 5C). [122] In this method, single-molecule m 6 A mapping was achieved by loading RNA sample to a coverslip coated with oligo-dT to ensure that only polyadenylated mRNA is captured. Captured mRNAs are then labelled at 3'-end by Cy3-dATP, and each labelled mRNA is individually registered using total internal reflection fluorescence (TIRF) microscopy.…”

Single-cell analysis of the epitranscriptome: RNA modifications under the microscope

“…It utilizes two proximity probes to target m6A-specific RNA and m6A methylation, followed by ligation and in situ rolling circle amplification (RCA) [ 68 ] Epitranscriptome profiling of microscopy-based, low-input samples and individual cells MUTZ3 leukaemia cells mRNAs from cell lysates on oligo-dT-coated coverslips are captured, individual m 6 A-immunobloated transcripts are visually detected and sequenced without being amplified. Finally, a nanoscale machine allows the isolation of individual cells, and thus, relate cell surface markers and gene expression signatures to single-cell m 6 A modification states [ 69 ] scDART-seq HEK293T cell line Deamination adjacent to RNA modification targets, cells expressing inducible APOBEC1-YTH [ 70 ] scm6A-seq In single oocytes/blastomeres of cleavage-stage embryos RNA fragmentation and ligation, labelling each twice and parallelized single-cell seq [ 71 ] picoMeRIP-seq In mouse embryonic stem (mES) cells and in single zebrafish zygotes, single mouse oocytes and preimplantation embryos RNA fragmentation, sonication, and RNA immunoprecipitation with validated anti-m 6 A [ 72 ] …”

“…In addition, a microscopy-based technology to measure gene expression, cell surface markers, and m 6 A modification in single cells and at single-molecule resolution was also developed [ 69 ]. Various innovations in nanowell technology were combined, together with low-quality digital gene expression (LQ-DGE), image registration, and sequential fluorescence in situ hybridization (seqFISH) to produce data containing many parameters from single cells.…”

One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N6-Methyladenosine Modifications

“…29 Although Kim et al developed a multimodal single-cell analysis platform, the application of microarrays strongly relies on specific algorithms and precise instruments. 30 The techniques described above still pose great challenges for single-cell RNA analysis. Hence, there is an urgent need to develop highly sensitive methods to detect and quantify specific m 6 A sites with high base resolution and isothermal conditions.…”

“…However, the method prefers to target the methylation (m 6 2 A) sites, and it is difficult to detect a single m 6 A site . Although Kim et al developed a multimodal single-cell analysis platform, the application of microarrays strongly relies on specific algorithms and precise instruments . The techniques described above still pose great challenges for single-cell RNA analysis.…”