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

ABSTRACT: 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 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. We 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.

SUBMITTER: Kim KL 

PROVIDER: S-EPMC8562683 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature