Improved Methods for Deamination-Based m6A Detection

Abstract

N ⁶-methyladenosine (m⁶A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m⁶A has been enabled by the identification of m⁶A sites transcriptome-wide. However, global m⁶A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m⁶A profiling from rare cell types or scarce tissue samples infeasible. To overcome this issue, we previously developed DART-seq, which relies on the expression of a fusion protein consisting of the APOBEC1 cytidine deaminase tethered to the m⁶A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m⁶A sites, therefore enabling single nucleotide-resolution m⁶A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m⁶A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m⁶A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m⁶A detection and will facilitate the mapping of m⁶A in samples not previously amenable to global m⁶A profiling.

Keywords: DART-seq; RNA biology; RNA modification; epitranscriptome; m6A.

FIGURE 1

Identification of an improved variant of the DART fusion protein. (A) Comparison of methylated RNAs identified by cellular DART-seq using expression of either APO1-YTH^D422N, APO1-YTH^DF1, or APO1-YTH^DF1(D401N) in HEK293T cells. Venn diagrams compare each DART protein variant to the original APO1-YTH protein. (B) Overlap of methylated RNAs identified by cellular DART-seq using the APO1-YTH^D422N protein with those identified by antibody-based methods. (C) Sanger sequencing traces showing C-to-U editing adjacent to m⁶A sites in cells expressing APO1-YTH^D422N, APO1-YTH, and APO1-YTH^mut for five mRNAs previously shown to contain m⁶A: DPM2, EIF4B, HERC2, NIPA1, and SMUG1. m⁶A sites are indicated by asterisks. C-to-U editing rate (%U) is indicated above the adjacent cytidine. Data are representative of three biological replicates. (D) Box plot showing the global C-to-U editing percentage of all sites common to HEK293T cells expressing APO1-YTH^D422N or APO1-YTH. (E) Western blot following RNA pulldown assays using purified DART proteins and bait RNAs. APO1-YTH^D422N exhibits improved binding to m⁶A compared to APO1-YTH.

FIGURE 2

ADARcd can be used as an alternative to APO1 to identify methylated RNAs with DART-seq. (A) Genome browser tracks showing two methylated mRNAs, AURKAIP1 and DPM2, in HEK293T cells expressing ADARcd-YTH^D422N, ADARcd-YTH^mut, or ADARcd alone. A-to-I editing found in at least 10% of the reads are indicated by red/blue coloring. m⁶A peaks identified by MeRIP (Meyer et al., 2012) is indicated in the bottom blue track. (B) Absolute distance plot showing the distance between A-to-I edit sites identified by ADARcd-YTH^D422N and m⁶A sites identified by miCLIP (Linder et al., 2015). (C) Metagene plot showing the distribution of A-to-I edit sites found in cells expressing ADARcd-YTH^D422N. (D) Venn diagram showing overlap between methylated RNAs identified by cellular DART-seq from HEK293T cells expressing ADARcd-YTH^D422N and methylated RNAs identified by antibody-based profiling (Meyer et al., 2012; Schwartz et al., 2014; Lichinchi et al., 2016). (E) Cumulative distribution plot (left) of %A-to-I for sites identified by ADARcd-YTH^D422N in untreated and STM2457 treated HEK293T cells. (F) Box plot showing the global A-to-I editing percentage of all sites common to both untreated and STEM2457 treated HEK293T cells expressing ADARcd-YTH^D422N. A Wilcoxon Rank-Sum test was conducted to access statistical significance.

FIGURE 3

In vitro DART-seq identifies m⁶A transcriptome-wide. (A) Comparison of C-to-U editing rates in methylated mRNAs obtained by in vitro DART-seq and cellular DART-seq. Sanger sequencing traces show C-to-U editing adjacent to m⁶A sites in a panel of five methylated mRNAs: DDX5, TUB, EIF4B, MKLN1, and HERC2. m⁶A sites are indicated by asterisks. C-to-U editing rate (%U) is indicated above the adjacent cytidine. Data Representative of three biological replicates. (B) Genome browser tracks of in vitro DART-seq data showing C-to-U editing in three representative mRNAs: ZZZ3, ATRX, and EEF1A1. C-to-U editing found in at least 10% of the reads is indicated by green/yellow coloring. m⁶A peaks identified by MeRIP (Meyer et al., 2012) is indicated in the bottom blue track. (C) Metagene analysis of m⁶A sites identified by in vitro DART-seq using the APO1-YTH^D422N protein. (D) Venn diagram showing the overlap between methylated RNAs identified by in vitro DART-seq filtered against the APO1-YTH^mut negative control and methylated RNAs identified by antibody-based methods (Meyer et al., 2012; Schwartz et al., 2014; Lichinchi et al., 2016). (E) Absolute distance plot showing the distance of C-to-U editing sites identified by in vitro DART-seq relative to m⁶A sites identified by miCLIP (Linder et al., 2015). m⁶A sites are centered at position 0. (F) Venn diagram showing the overlap between methylated RNAs identified by in vitro DART-seq compared to methylated RNAs found by cellular DART-seq.

FIGURE 4

Blocking with the YTH domain minimizes false positives with in vitro DART-seq. (A) Metagene analysis of C-to-U editing sites in mRNAs identified with in vitro DART-seq using the APO1-YTH^D422N protein after pre-incubation with the YTH domain (left) or the APO1-YTH^mut protein (right). (B) Venn diagram showing the overlap between methylated RNAs identified by in vitro DART-seq with APO1-YTH^D422N filtered by YTH blocking and methylated RNAs identified by antibody-based methods (Meyer et al., 2012; Schwartz et al., 2014; Lichinchi et al., 2016). (C) Metagene analysis showing the distribution of C-to-U editing sites in mRNAs after filtering of in vitro DART-seq data against by YTH blocking (blue) or by APO1-YTH^mut (red). (D) Venn diagram of C-to-U edit sites induced by in vitro DART-seq with APO1-YTH^D422N, filtered by use of either YTH blocking or APO1-YTH^mut (left). Venn diagram of methylated RNA identified by in vitro DART-seq with APO1-YTH^D422N, filtered by use of either YTH blocking or APO1-YTH^mut (right).