ScDART-Seq Uncovers Diverse mRNA Methylation Patterns in Single Cells
Summary
N6-methyladenosine (m6A), a prevalent RNA modification, serves as a crucial regulator of RNA and cellular function. While m6A distribution and function have been explored extensively in large cell populations, the application of such studies has remained limited to date. In this study, a novel approach is proposed to identify transcriptome-wide m6A sites within individual cells. Surprisingly, substantial heterogeneity in the abundance and presence of m6A sites across cells is discovered. Differential methylation of mRNAs across the cell cycle is also identified, and RNA methylation signatures enable the differentiation of cellular subpopulations independently of gene expression. These findings reveal fundamental features of m6A that were previously overlooked through bulk cell profiling and suggest the existence of cell-intrinsic mechanisms responsible for m6A deposition.
Fig.1 Graphical abstract. (Tegowski, 2022)
Research Criteria
The research idea of this article is to develop a method for transcriptome-wide profiling of m6A sites in single cells. The authors use a strategy called DART-seq that leverages an enzyme called APOBEC1 and a protein domain called YTH to label and capture m6A sites in RNA. They apply this method to thousands of individual cells and uncover surprising heterogeneity and variability in the presence and abundance of m6A sites across different cells and transcripts. They also identify differentially methylated RNAs across the cell cycle and cellular subpopulations. These findings reveal fundamental features of m6A that have been missed by bulk cell profiling and suggest the existence of cell-intrinsic mechanisms for m6A deposition.
Sample Type
Human cell line.
Result—scDART-seq Identifies m6A Sites in Single Cells
The authors employed the APOBEC1-YTH expression method and performed drop-based single-cell RNA sequencing (scRNA-seq) on a total of 10,352 individual cells in three biological replicates to identify m6A sites at the single-cell level. The Bullseye tool was then utilized to pinpoint m6A sites in single cells, which revealed 16,934 high-confidence sites in 3,844 RNAs across all 10,352 cells. Importantly, the detection of m6A sites and methylated RNAs was highly consistent across replicates, indicating the robustness and reproducibility of the scDART-seq approach. Remarkably, the identified single-cell m6A sites exhibit similar characteristics to those observed at the population level, including a significant enrichment in the vicinity of the stop codon and in the RAC (R=A/G) consensus sequence. Additionally, the authors observed a high degree of overlap between the methylated RNAs identified using scDART-seq and those identified in bulk cells through antibody-based m6A mapping. Taken together, these data highlight the capability of scDART-seq to accurately and reproducibly identify m6A sites in single cells.
Fig.2 scDART-seq detects m6A in single cells. (Tegowski, 2022)
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Reference
- Tegowski, M.; et al. scDART-Seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells. Molecular Cell. 2022, 82(4): 868-870.
