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

Bradley E Bernstein,Daniel R Jones,Ekaterina N Andreishcheva,Efrat Shema,Volker Hovestadt,Abhijeet Shinde,Kyung Lock Kim,Elizabeth Gaskell,Peter Van Galen,Gilbert J Rahme

doi:10.1016/j.crmeth.2021.100061

Abstract

SummaryEpigenetic 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.

Highlights

Chemical modifications of mRNA regulate transcript and protein abundance, thereby affecting cellular state
We developed a microscopy-based platform to measure cell surface markers, gene expression, and m6A levels in individual cells and at single-molecule resolution
We began by redesigning low-quantity digital gene expression (LQ-DGE) technology, which combines sequential base additions with single-molecule total internal reflection fluorescence (TIRF) imaging (Ozsolak et al, 2009, 2010)

Summary

Introduction

Chemical modifications of mRNA regulate transcript and protein abundance, thereby affecting cellular state. The most abundant of these modifications is m6A, the levels of which vary widely between cell types and states, and 20%–40% of all mRNAs contain one or more m6A modifications (Dominissini et al, 2012; Frye et al, 2018; Meyer et al, 2012). Antibody-based methods, such as m6A sequencing, MeRIP sequencing, and m6A-LAIC sequencing (m6A-LAIC-seq), provided the first transcriptome-wide view of m6A but require large amounts of input RNA (1~3 μg) (Dominissini et al, 2012; Meyer et al, 2012; Molinie et al, 2016). To build quantitative models of the impact of mRNA modifications on gene expression and cellular state, we require technologies that can quantify RNA modifications and transcript abundance–and ideally other measures of cellular state, such as surface markers–within single cells

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Discussion

Conclusion