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

Figures & data

Figure 1. RNA modifications detected and those yet to be found. Better sequencing technologies have allowed the identification of millions of new modification sites in all types of RNAs. Considering that > 170 different types of modified ribonucleotides have been found until now [9], it is to be expected that these novel technologies will result in a big increase in identifiable RNA modifications which may be now hidden below the detection threshold. Created with BioRender.com [10].

Figure 2. Biological effects of m6A modification in human mRNA depending on its position. Depending on where the modification m6A is added onto the mRNA molecule it may enhance different consequences, such as translation, splicing, its exportation or even an increase of the molecule’s instability. Created with BioRender.com [10].

Figure 3. Single-cell m6A sequencing technologies. From the top in a clockwise manner, small explanation of all existing techniques: m6AISH-PLA, epitranscriptome profiler, scDART-seq, scm6A-seq and picoMeRIP-seq. Created with BioRender.com [10].

Table 1. List of single-cell technologies being used to study m⁶A RNA modification, with the correspondent cell types it was used on and a small explanation of the various methodologies.

Single-cell methods used to study RNA modification: m^6A
Method name	Single cells used on	Method	Ref.
m^6AISH-PLA	Hepa 1–6 Cell Line	m^6A-specific in situ hybridization mediated proximity ligation assay for cellular imaging of m^6A RNA. 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^6A-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^6A 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^6A	[72]

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