Epigenetic modifications control the stability and translation of mRNA molecules. Researchers from Massachusetts General Hospital and Harvard Medical School have developed a microscopy-based platform for quantifying modified RNA molecules and for relating the modification patterns to single-cell phenotypes. The researchers directly capture mRNAs from cell lysates on oligo-dT-coated coverslips, then visually detect and sequence individual m6A-immunolabled transcripts without amplification. Integration of a nanoscale device enabled us to isolate single cells on the platform, and thereby relate single-cell m6A modification states to gene expression signatures and cell surface markers. Application of the platform to MUTZ3 leukemia cells revealed a marked reduction in cellular m6A levels as CD34+ leukemic progenitors differentiate to CD14+ myeloid cells. The researchers then coupled single-molecule m6A detection with fluorescence in situ hybridization (FISH) to relate mRNA and m6A 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.
Transcriptome-wide m6A profiling at single-molecule resolution
(A) LQ-DGE with m6A detection. PolyA+ RNA from ~1,000 GM12878 cells was captured on an oligo-dT-coated coverslip followed by antibody detection of m6A. Single-molecule sequencing of all transcripts was then performed by reverse transcription followed by second-strand cDNA synthesis. TdT, terminal deoxynucleotidyl transferase (B) Dot blot assay with in vitro synthesized m6A−/+ transcripts (in vitro generated transcripts [IVTs]) using anti-m6A antibody. m6A− IVTs were unmodified, and m6A+ IVTs contained an average of 12 m6ATP nucleotides per transcript. (C) TIRF microscopy images showing m6A− or m6A+ Cy3-labeled IVTs (green) stained with an anti-m6A antibody and an Alexa Fluor 647-conjugated secondary antibody (red). Scale bar, 5 μm. (D) Quantification of m6A detection rates by analyzing colocalization of anti-m6A antibody and Cy3 fluorescence signals. (E) Scatterplot showing the correlation between modified LQ-DGE (0.51 M reads) and RNA sequencing (RNA-seq) (50 M reads) data for GM 12878 cells. (F) Scatterplot showing the correlation between gene-specific m6A levels from the LQ-DGE (total, 0.51 M reads and m6A+, 0.14 M reads) and those from m6A-LAIC-seq (m6A-negative or m6A-positive sample, each 50 M reads).