High-resolution N(6) -methyladenosine (m(6) A) map using photo-crosslinking-assisted m(6) A sequencing

Abstract

N(6) -methyladenosine (m(6) A) is an abundant internal modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism. However, methods to precisely locate the m(6) A modification remain limited. We present here a photo-crosslinking-assisted m(6) A sequencing strategy (PA-m(6) A-seq) to more accurately define sites with m(6) A modification. Using this strategy, we obtained a high-resolution map of m(6) A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m(6) A sites at base resolution. Our results provide insight into the relationship between the methylation regions and the binding sites of RNA-binding proteins.

Keywords: N6-methyladenosine; RNA modification; photo-crosslinking; transcriptome sequencing.

Figure 1

Model study using PA-m⁶A-seq. a) Control study to confirm crosslinking is based on recognition of antibody against m⁶A-containing RNA. b) Control study to prove UV365 is the trigger of covalent crosslinking by using a synthesized 21-mer RNA oligonucleotide. c) T-to-C transition introduced by 4SU and UV irradiation. Proposed mechanism on how the 4SU crosslinked with protein changes the Watson–Crick base pair (top). The sequence of 21-mer model is shown in the middle. Sanger sequencing results of 21-mer model with and without UV365 proved the crosslinked 4SU near m⁶A was read as C. The blue arrows indicate the 4SU sites on the model, whereas red arrows point out the chromatogram reads of corresponding 4SU with or without crosslinking.

Figure 2

PA-m⁶A-seq applied to poly(A)-tailed RNA purified from HeLa cells. In following figures, blue bars represent methylation sites identified by PA-m⁶A-seq, whereas blue ‘peaks’ above those bars are from normal m⁶A-seq. a) Validation of PA-m⁶A-seq strategy. Metagene profile and pie-chart of the enrichment of RNA segments are consistent with previous reported distribution of m⁶A, and the motif search yielded GGACU as the predominant one, which was the same as the result from normal m⁶A-seq. b) Comparison of predicted methylation sites in β-actin (ACTB) and homo sapien basigin (BSG) from PA-m⁶A-seq with peaks from normal m⁶A-seq and single sites by SCARLET. Sequences of predicted sites are shown below, consensus motif containing m⁶A in red. All input background of normal m⁶A-seq has been subtracted. Both sites were verified by SCARLET. c) Multiple methylation sites in MALAT1 transcript. The methylation sites confirmed by SCARLET, which were covered by peaks from normal m⁶A-seq, were also identified by PA-m⁶A-seq with higher resolution. Yellow regions indicate the RRACH motif. Red lines are the probes used to verify these sites with SCARLET. d) Distance of predicted methylation sites using PA-m⁶A-seq versus peaks obtained from normal m⁶A-seq to YTHDF2-binding sites. e) Distance of predicted methylation sites obtained from PA-m⁶A-seq versus peaks obtained from normal m⁶A-seq to HuR-binding sites.

Scheme 1

The strategy of photo-crosslinking-assisted m⁶A-seq (PA-m⁶A-seq). Covalently crosslinked 4SU is labeled as U*, which is read as C in RT-PCR. The example of the high-throughput sequencing result is shown on the bottom right. Black bold vertical bars represent T-to-C transition induced by 4SU and covalent crosslinking, compared to reference genome hg19.