Evolutionarily conserved human targets of adenosine to inosine RNA editing

Abstract

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice.

Figure 1

Editing in FLNA transcripts. (a) Some of the publicly available expressed sequences covering this gene, together with the corresponding genomic sequence. A total of 226 sequences are available for this locus, 23 of which are edited. (b) Results of sequencing experiments. Matching human DNA and cDNA RNA sequences for human brain and lung tissues. Editing is characterized by a trace of guanosine in the cDNA RNA sequence, where the DNA sequence exhibits only adenosine signals. Sequencing data for more tissues are available as Supplementary Material. Note the variety of tissues showing editing and the variance in the relative intensity of the edited guanosine signal. (c) Sequences of individually cloned fragments from matching DNA and RNA of mouse brain tissues and chicken brain and liver tissues. Only part of the data is shown. A total of 20 mouse brain cDNA clones, 10 chicken brain and 9 chicken liver cDNA clones were sequenced, out of which four, seven and one sequence showed editing events, respectively. Similar results for the other two substrates are provided as Supplementary Material.

Figure 2

Editing in CYFIP2 transcripts. (a) Some of the publicly available expressed sequences covering this gene, together with the corresponding genomic sequence. A total of 23 sequences are available for this locus, two of which are edited. Both edited sequences originate from brain tissues. (b) Results of sequencing experiments. Matching human DNA and cDNA sequences for human brain and prostate cDNA. As in Figure 1, editing is characterized by a trace of guanosine in the cDNA RNA sequence, where the DNA sequence exhibits only adenosine signals. Sequencing data for more tissues are available in Supplementary Figure. In the brain, the editing signal surpasses the original adenosine signal, but in other tissues it is very weak. (c) Sequences of individually cloned fragments from matching DNA and RNA of mouse brain tissues and chicken brain and liver tissues. Only part of the data is shown. A total of eight mouse brain cDNA clones were sequenced and all of them were edited. Nine chicken brain cDNA clones were sequenced, out of which four were edited. In contrast, none of the eight chicken liver cDNA clones was edited. These results suggest that editing of this site might be brain specific, in agreement with the data for human tissues presented in the previous panel. Similar results for the other two substrates are provided as Supplementary Material.

Figure 3

Hairpin structure in BLCAP transcripts. (a) The predicted secondary structure for the BLCAP substrate, based on lowest free-energy predictions using the program MFOLD (50) (). The editing site is at position 601, where the codon UAU(Y) is edited into UGU(C). Structures for the other substrates are given in Supplementary Material. (b) Conservation levels at the editing genomic locus. The two red bars at the bottom mark the editing region and the intronic sequence almost perfectly pairing with it to form the hairpin structure shown in (a). The editing site is marked in black within the left red bar. The high conservation level of the intronic sequence, suggesting a functional importance, supports its identification as necessary for the editing process.

Figure 4

Ribbon representation of the molecular model of alpha-filamin repeat 22 generated using the Ig-like repeat from ABP120 as the template. The edited residue Gln is highlighted.