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. 2014 Mar;24(3):522-34.
doi: 10.1101/gr.162537.113. Epub 2014 Jan 9.

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome

Masayuki Sakurai  1 , Hiroki Ueda, Takanori Yano, Shunpei Okada, Hideki Terajima, Toutai Mitsuyama, Atsushi Toyoda, Asao Fujiyama, Hitomi Kawabata, Tsutomu Suzuki
Affiliations

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome

Masayuki Sakurai et al. Genome Res. 2014 Mar.

Abstract

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called "inosine chemical erasing" (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions.

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Figures

Figure 1.
Figure 1.
Biochemical identification of A-to-I editing sites by ICE-seq. (A) Chemistry of inosine cyanoethylation. Acrylonitrile adducts to the N1 position of inosine to form N1-cyanoethylinosine (ce1I). (B) Outline of ICE-seq. Schemes without (CE−) or with (CE+ or CE++) cyanoethylation of RNA are shown on the left and right, respectively. RNA and cDNA are indicated by gray and black arrows, respectively. (i) Cyanoethylation and fragmentation. The I in the RNA strand is specifically cyanoethylated to form ce1I (CE+). RNAs are partially digested by mild alkaline treatment. (ii) First strand cDNA synthesis. RNAs are reverse-transcribed with a random primer. RNA bearing an A at the editing site is converted to T in the cDNA in both conditions (CE− or CE+). In the CE− condition, RNA bearing I is transcribed to C in the cDNA. In the CE+ condition, first strand cDNA extension is arrested at the ce1I site (red arrow). (iii) Second strands are synthesized to obtain double-stranded cDNAs which are then subjected to the end-repair reaction and adaptor ligation. (iv) Gel purification of 300-bp cDNAs for PCR amplification. The amplified cDNAs with 300 bp are gel-purified again. In this step, cDNAs arrested at ce1I are discarded. (v) The cDNAs for CE−, CE+, and CE++ conditions are sequenced from both ends by a GA2 sequencer. Data processing of these reads identifies inosines by detecting erased reads upon cyanoethylation.
Figure 2.
Figure 2.
Sequence statistics of ICE-seq. (A) Numbers of mapped tags used for ICE-seq in CE−, CE+, and CE++ conditions. The reference genome and cDNA sequences used here are hg18 and the UCSC gene, respectively. (B) Distribution histogram of gene expression estimated by the read coverage. Gene expression represented by the Log2(RPKM) value for the reference transcriptome sequence is compiled at 0.1 Log2(RPKM) intervals under conditions of CE− (blue), CE+ (yellow), and CE++ (magenta). (C) Pie chart of gene expression estimated by the read coverage. A total of 63% of human genes are detected with more than 20 times coverage (∼1 RPKM). (D) Scatter plots of logarithmic RPKM values in CE− versus CE+ or CE++. Their coefficients of determination (R2) are 0.998 and 0.995, respectively.
Figure 3.
Figure 3.
Genome-wide views of the regions with editing sites piled with the mapped reads of ICE-seq. (A) Q/R and Q/Q sites in exon 11 of GRIA2 mRNA. (B) I/M site in GABRA3 mRNA. (C) Editing cluster in 3′ UTR of BPNT1 mRNA. Top panel shows histograms of the mapped reads under CE− conditions at genomic positions. Genome number, positions, and scale of length are indicated. Middle panel shows close-up views of the regions with editing sites piled with the mapped reads in conditions of CE− (blue), CE+ (orange), and CE++ (pink). Bottom panel shows the decreased read ratio upon cyanoethylation (CE++) (gray downward peaks) and editing sites with the decreased ratio of G-base counts upon cyanoethylation (CE++) (red bars).
Figure 4.
Figure 4.
Separation of A-to-I editing sites from false-positive sites and SNPs. (A) Scatter plot of ΔNg(CE+) versus ΔNg(CE++). The 40 known editing sites in CDS (Supplemental Table S3) and 1963 editing sites validated by the ICE method in non-CDS (Supplemental Table S4) are indicated by light blue and magenta points, respectively. cSNPs and gSNPs are indicated by yellow and orange points, respectively. Dark blue points are nonannotated A-to-G sites. (B) Scatter plot of ΔNn(CE+) versus ΔNn(CE++). cSNPs and gSNPs are shown as yellow and orange points, respectively. Dark blue points are nonannotated mismatched sites. (C) Histogram of the ICE scores for A-to-G sites from SNPs (yellow), known editing sites (magenta), and nonannotated sites (blue). (D) Histogram of the ICE scores for non-A-to-G sites from SNPs (yellow) and nonannotated sites (blue).
Figure 5.
Figure 5.
Statistical features of A-to-I editing sites detected by ICE-seq. (A) Venn diagrams show the number of novel editing sites detected by ICE-seq and the ICE method in this study (left panel), both from known and novel editing sites detected in this study (middle panel), and the number of editing sites detected by us and known/predicted sites deposited in DARNED (right panel). (B) Plot of the Gr value versus the G ratio at each editing site. The coefficient of determination (R2) is 0.77. (C) Editing frequency distribution of the sites detected by ICE-seq. The editing frequency of each site was calculated from the Gr value. The numbers of editing sites at each template ratio are compiled in the histogram. Gray and white boxes represent novel and known/predicted sites, respectively. (D) Base preference around the editing site presented by WebLogo using the full ICE-seq data set (left) and sites with >50% editing frequency (right). (E) Triplet preference of editing. Statistics of triplet sequences centered on the edited A were analyzed using the full ICE-seq data set (left) and sites with >50% editing frequency (right).
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