Widespread cleavage of A-to-I hyperediting substrates

Abstract

A-to-I RNA editing is the conversion of adenosine to inosine in double-stranded cellular and viral RNAs. Recently, abundant hyperediting of human transcripts, affecting thousands of genes, has been reported. Most of these editing sites are confined to intramolecular hairpin double-stranded RNA (dsRNA) structures formed by pairing of neighboring, reversely oriented, primate-specific Alu repeats. The biological implication of this extensive modification is still a mystery. A number of studies have shown that heavily edited transcripts are often retained in the nucleus. A recent study found that the edited region in transcripts of the mouse Slc7a2 gene is post-transcriptionally cleaved upon stress, enabling the release of the mRNA to the cytoplasm, followed by its translation. Here, we aim to test whether this scenario might be relevant for many other hyperedited Alu targets. Bioinformatics analysis of publicly available mRNA and expressed sequence tag data provides evidence showing that neighboring, reversely oriented, Alu elements are often cleaved at both ends of the region harboring the inverted repeats followed by rejoining of the two parts of the transcript on both sides of the inverted repeats, resulting in almost inosine-free mRNA products. Deleted segments vary among transcripts of the same gene and are not flanked by the canonical splicing signal sequences. The tissue distribution of these events seems to correlate with known A-to-I editing patterns, suggesting that it depends on the dsRNA structure being edited. Results are experimentally verified by polymerase chain reaction and cloning data. A database of 566 human and 107 mouse putative cleavage loci is supplied.

FIGURE 1.

UCSC alignment of mRNAs with the 3′ UTR of the apolipoprotein L1 isoform b precursor (APOL1) transcript. (Top panel) Reference sequences for this gene going (left to right) through the end of the last intron (arrowed line), the end of the coding sequence (thick bar), and the 3′ UTR (thin bar). (Middle panel) Different mRNA sequences supporting this region. Most of these sequences (11 out of 15) show signatures of cleavage—a missing part in the middle of the 3′ UTR, looking like an intron (arrowed line). Ten out of the 11 were identified by the present algorithm, while the 11th (CR626091) happened to be cleaved at a canonical splicing signal site. (Bottom panel) Location of the Alu repeats. Unlike splicing, the exact cleavage sites differ between different mRNAs, but they are located within the inverted Alus, or close to their border, in all cases.

FIGURE 2.

Deletion of hyperedited inverted Alu repeat structures within the 3′ UTRs of METTL7A and PSMD12 transcripts. PCR performed on normal human hippocampus genomic DNA using primers flanking the inverted repeat structure (MF1+MR for METTL7A; PF1+PR for PSMD12) results in the expected full-length amplicon (lane 1). However, using the same primers but cDNA from the same tissue as a template, one observes a band of much shorter length, corresponding to completely/partially cleaved isoforms (lane 2). PCR performed on cDNA using an internal primer (MF2+MR for METTL7A; PF2+PR for PSMD12) demonstrates the coexistence of mRNA isoforms with complete 3′ UTRs in which the NCIs were not removed (lane 3). These longer isoforms are not apparent in lane 2 owing to differences in reaction efficiencies.