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. 2011 Feb;18(2):230-6.
doi: 10.1038/nsmb.1975. Epub 2011 Jan 23.

Transcriptome-wide sequencing reveals numerous APOBEC1 mRNA-editing targets in transcript 3' UTRs

Brad R Rosenberg  1 , Claire E Hamilton, Michael M Mwangi, Scott Dewell, F Nina Papavasiliou
Affiliations

Transcriptome-wide sequencing reveals numerous APOBEC1 mRNA-editing targets in transcript 3' UTRs

Brad R Rosenberg et al. Nat Struct Mol Biol. 2011 Feb.

Erratum in

  • Nat Struct Mol Biol. 2012 Mar;19(3):364

Abstract

Apolipoprotein B-editing enzyme, catalytic polypeptide-1 (APOBEC1) is a cytidine deaminase initially identified by its activity in converting a specific cytidine (C) to uridine (U) in apolipoprotein B (apoB) mRNA transcripts in the small intestine. Editing results in the translation of a truncated apoB isoform with distinct functions in lipid transport. To address the possibility that APOBEC1 edits additional mRNAs, we developed a transcriptome-wide comparative RNA sequencing (RNA-Seq) screen. We identified and validated 32 previously undescribed mRNA targets of APOBEC1 editing, all of which are located in AU-rich segments of transcript 3' untranslated regions (3' UTRs). Further analysis established several characteristic sequence features of editing targets, which were predictive for the identification of additional APOBEC1 substrates. The transcriptomics approach to RNA editing presented here dramatically expands the list of APOBEC1 mRNA editing targets and reveals a novel cellular mechanism for the modification of transcript 3' UTRs.

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Figures

FIGURE 1
FIGURE 1
Comparative RNA-Seq Screen for APOBEC1 mRNA Editing Targets. Schematic workflow of RNA-Seq Screen.
FIGURE 2
FIGURE 2
Validation of APOBEC1 mRNA Editing Targets. (a, b, c and d) Representative examples of conventional Sanger sequencing chromatograms for wild-type and Apobec1−/− genomic DNA and cDNA at editing sites. (a) chr4:57203753(−) in the Ptpn3 transcript, (b) chr16:43981376(−) in the Gramd1c transcript, (c) chr2:121978638(+) in the B2m transcript, and (d) chr3:73442586(−) in the Bche transcript. (e) Editing frequency of ranked APOBEC1 sites. ‡ indicates apoB editing site.
FIGURE 3
FIGURE 3
Sequence Features of APOBEC1 Editing Sites. (a) AU content of random sets of 3′ UTRs. The value for the APOBEC1 editing site-containing 3′ UTRs is well to the right of the distribution. (b) AU content of random 101-nt windows within APOBEC1 editing site-containing 3′ UTRs. The value for the windows centered on the editing sites is well to the right of the distribution. (c) AU content in a sliding 101-nt window in the Tmbim6 3′ UTR. The AU content peaks near the editing site, chr15:99239051(+). (d) Frequency plot of bases flanking the APOBEC1 editing sites, aligned on the target cytidine. (e) Base counts of nucleotides flanking the APOBEC1 editing sites. The nucleotides immediately adjacent to the target cytidine tend overwhelmingly to be A or U. P values were computed using the binomial test. For a given column, the P value is the probability that the skew towards A or U is a random occurrence.
FIGURE 4
FIGURE 4
APOBEC1 mRNA Editing Targets Share a Characteristic Sequence Motif. (a) Frequency plot of sequence motif identified by MEME analysis of regions flanking APOBEC1 editing site. Log likelihood ratio (157) and E value (8.8 ×10−1) are significant as compared to the “best” motif of a shuffled sequence control (Log likelihood ratio 65, E value 3 × 102). (b) Alignment of APOBEC1 target sequences by consensus sequence motif. Edited cytidines are shaded in blue. Yellow shading indicates a match to the consensus sequence motif, represented in green.
FIGURE 5
FIGURE 5
Sequence Pattern Prediction of APOBEC1 mRNA Editing Targets. (a) APOBEC1 editing site pattern used to search for additional targets in RefSeq transcripts. (b) Occurrences of APOBEC1 editing site pattern in RefSeq transcripts by type, listed by intestine epithelium expression level and wild-type RNA-Seq read coverage. (c) Editing frequency at predicted APOBEC1 target sites as evaluated by wild-type read content. ‡ indicates apoB editing site. (d) Sanger sequencing validation of predicted APOBEC1 editing sites in RefSeq transcripts by type. (e and f) Representative examples of conventional Sanger sequencing chromatograms for wild-type and Apobec1−/− genomic DNA and cDNA at predicted editing sites. (e) chrX:101478733(−) in the Abcb7 transcript, (f) chr9:114658289(+) in the Cmtm6 transcript.
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