DART-seq: an antibody-free method for global m6A detection

2019-10-06 by Quick Biology Inc.

Modification to RNA species (Fig 1) has been discovered for over 50 years.  The internal modification m⁶A N⁶-methyladenosine (m⁶A) is the most abundant type, plays diverse roles in a variety of physiological processes.

To date, most global m⁶A detection relies on RNA immunoprecipitation by m⁶A-recognizing antibodies, such as MeRIP-seq, m⁶A-seq. These methods are time-consuming and costly (expensive antibody, a large amount of input RNA). In recently Nature Methods, Dr. Meyer developed DART-seq:  an antibody-free method for global m⁶A detection. In this experiment, Dr. Meyer fused APOBEC1 to the m⁶A-binding YTH domain. Recruitment of APOBEC1 to m⁶A site will enable deamination of cytidine immediately following m⁶A residue like C-to-U editing. APOBEC1-YTH is transiently expressed in cells and total RNA is isolated and subjected to RNA-seq. C-to-U mutations are then detected to identify sites of m⁶A (Fig 2).

To validate DARP-seq approach, the author performed DART-seq, compared to m⁶A sites discovered previously by miCLIP (Fig 3).  To rule out any potential cell alteration caused by APOBEC1-YTH transfection in cell, she also performed in vitro DART-seq just using HEK293T cell RNA treated by APOBEC1-YTH protein (Fig 4).  Both data show great agreement with traditional antibody-based methods.

Figure 1: Chemical modification in eukaryotic messenger RNAs (ref1). A schematic representation of common chemical modifications in eukaryotic mRNA transcripts. Several of these modifications map uniquely to the mRNA cap structure, 5’ or 3’ untranslated regions, or the coding region (bold) of the transcript.

Figure 2: Schematic of DART-seq (ref2). APOBEC1 is fused to the YTH domain to guide C-to-U editing at cytidine residues adjacent to m6A sites. APOBEC1-YTH is expressed in cells and total RNA is isolated and subjected to RNA-seq. C-to-U mutations are then detected to identify sites of m6A.

Figure 3: Example of m6A sites detected by DART-seq (ref2). C-to-U mutations found in at least 10% of reads are indicated by gold/green coloring at individual sites (gold indicates the abundance of C sites and green indicates the abundance of U sites at each position; ACTB is transcribed from the negative strand). Percentages of C-to-U mutations at two individual sites previously identified by miCLIP5 are indicated in the expanded view. Results are representative of three independent experiments.

Figure 4:  In Vitro DART-seq identifies m6A sites in cellular RNAs. (ref2)

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Reference:

1. 1. Roundtree etc Dynamic RNA modifications in gene expression regulation. Cell. 169, 1187–1200
2. 2. Meyer, K. D. DART-seq: an antibody-free method for global m6A detection. Nat. Methods (2019). doi:10.1038/s41592-019-0570-0 (2017).