A survey of RNA editing in human brain

Abstract

We have conducted a survey of RNA editing in human brain by comparing sequences of clones from a human brain cDNA library to the reference human genome sequence and to genomic DNA from the same individual. In the RNA sample from which the library was constructed, approximately 1:2000 nucleotides were edited out of >3 Mb surveyed. All edits were adenosine to inosine (A-->I) and were predominantly in intronic and in intergenic RNAs. No edits were found in translated exons and few in untranslated exons. Most edits were in high-copy-number repeats, usually Alus. Analysis of the genome in the vicinity of edited sequences strongly supports the idea that formation of intramolecular double-stranded RNA with an inverted copy underlies most A-->I editing. The likelihood of editing is increased by the presence of two inverted copies of a sequence within the same intron, proximity of the two sequences to each other (preferably within 2 kb), and by a high density of inverted copies in the vicinity. Editing exhibits sequence preferences and is less likely at an adenosine 3' to a guanosine and more likely at an adenosine 5' to a guanosine. Simulation by BLAST alignment of the double-stranded RNA molecules that underlie known edits indicates that there is a greater likelihood of A-->I editing at A:C mismatches than editing at other mismatches or at A:U matches. However, because A:U matches in double-stranded RNA are more common than all mismatches, overall the likely effect of editing is to increase the number of mismatches in double-stranded RNA.

Figure 1.

Proportion of edited and unedited Alus with additional Alus in the same intron. All Alus aligning to the introns of known genes, and for which ≥80% of the genomic extent of the Alu was sequenced, were included in this analysis. The proportion of edited Alus (gray bars) and the proportion of unedited Alus (black bars) having an antisense Alu (A) or a same sense Alu (B) in the same intron are shown for different intron sizes.

Figure 2.

Distance from edited and unedited Alus to the nearest Alu in the same intron. The Alu sequences included in this analysis are the same as in Figure 1. The proportion of edited Alus (gray bars) and the proportion of unedited Alus (black bars) at different distances from the nearest antisense Alu (A) or same sense Alu (B) are shown.

Figure 3.

Amount of flanking Alu sequence at different distances from edited and unedited Alus. All Alus for which ≥80% of the genomic extent of the Alu was sequenced were included in this analysis. For each Alu, the amount of flanking Alu sequence in the opposite orientation (A) or same orientation (B) in successive 1-kb windows was recorded. For each distance, the flanking Alu sequences in the 1-kb window 5′ and 3′ of the reference Alu were combined. The data presented are the average amount of Alu sequence flanking all edited Alus (gray bars) or unedited Alus (black bars).

Figure 4.

Sequence context of adenosines in edited Alu sequences. The sequence context of all edited adenosines and all unedited adenosines from all edited Alu sequences was compared. For each of the 10 bases either side of edited adenosines (dashed lines) and unedited adenosines (solid lines), the proportion of bases that were A, C, G, or T (A-D, respectively) at that position was calculated.

Figure 5.

Effect of sequence composition on the likelihood of RNA editing. A multiple alignment of all edited Alu sequences was prepared using CLUSTALW. At each position in the alignment, the proportion of edited adenosines was calculated from the number of sequenced edited adenosines and the total number of sequenced adenosines. The sequence composition at each position was calculated from all Alus. For each position in the alignment, the proportion of edited adenosines is compared to the proportion of A, C, G, or T at that position (A-D, respectively).