A method to find tissue-specific novel sites of selective adenosine deamination

Abstract

Site-selective adenosine (A) to inosine (I) RNA editing by the ADAR enzymes has been found in a variety of metazoan from fly to human. Here we describe a method to detect novel site-selective A to I editing that can be used on various tissues as well as species. We have shown previously that there is a preference for ADAR2-binding to selectively edited sites over non-specific interactions with random sequences of double-stranded RNA. The method utilizes immunoprecipitation (IP) of intrinsic RNA-protein complexes to extract substrates subjected to site-selective editing in vivo, in combination with microarray analyses of the captured RNAs. We show that known single sites of A to I editing can be detected after IP using an antibody against the ADAR2 protein. The RNA substrates were verified by RT-PCR, RNase protection and microarray. Using this method it is possible to uniquely identify novel single sites of selective A to I editing.

Figure 1

Illustration of the IP-array method to find novel substrates for A to I editing. Cell lysis extract was prepared from mouse brain. The extract was immunoprecipitated using an ADAR2-specific polyclonal antibody. Target RNA was extracted from the mRNP complexes upon protein removal. The RNA was amplified, labeled and further hybridized to a mouse genomic oligo array.

Figure 2

Western blot analysis using anti-human ADAR2 antibody on IP eluates. Three mouse brains were used for ADAR2-specific and pre-immune serum IP, respectively. One-tenth of the IP eluate was used for western blot. Lane 1 represents the pre-immune serum IP and lane 2 shows the amount of ADAR2 in the ADAR2-specific IP.

Figure 3

Detection of known substrates for A to I editing using ADAR2-specific IP. (a) Semi-quantitative RT–PCR on GluR-B at the R/G and Q/R site, GluR-A and the serotonin receptor (5-HT_2C) using radioactively labeled primers. Lane 1 shows the amplification of the R/G site from an ADAR2-specific IP, with an estimated size of 314 bp. Lane 2 shows a product amplified from the R/G site from an IP using pre-immune serum. Lane 3 shows the amplification of the Q/R site from an ADAR2-specific IP, with an estimated size of 253 bp. Lane 4 shows product amplification of the Q/R site from an IP using pre-immune serum. RT–PCR on GluR-A, lacking sites for A to I editing, shows no detectable amplification from an ADAR2-specific IP (lane 5), or a pre-immune serum IP (lane 6). Product using total RNA is shown in lane 7 and the estimated size is 203 bp. RT–PCR on 5-HT_2C shows the amplification from an ADAR2-specific IP (lane 8), the estimated size is 94 bp. Lane 9 shows a product amplified from the 5-HT_2C transcript from an IP using pre-immune serum. Lane M is a size marker with bands of sizes as indicated. (b) RT–PCR on the ribosomal phosphoprotein P0, lacking sites for A to I editing. No enrichment could be detected in the ADAR2 IP (lane 1) compared with the pre-immune serum IP (lane 2). The estimated size is 265 bp. Lane M is a size marker with bands of sizes as indicated. (c) The product from the RT–PCR-specific for the R/G and the Q/R sites were DNA sequenced to determine the editing efficiency. At the R/G site a forward primer was used to give a dual A and G peak at the R/G site. At the Q/R site a reverse primer was used so that an edited site is a C in the sequence. Edited nucleotides are indicated with an arrow in the chromatogram. (d) An RNase protection assay was used to confirm the enrichment of GluR-B in the presence of anti-ADAR2 antibody. A 225 nt long α-³²P-labeled probe, 118 nt complementary downstream of the R/G site, was hybridized to RNA from an IP using pre-immune serum (lane 1) and to RNA from an IP using anti-ADAR2 antibody (lane 2).

Figure 4

Genes enriched in ADAR2 IP compared with pre-immune serum IP. 200 genes were significantly increased in all three different arrays. The mean value for the three arrays is shown as fold increase 2^x. The GluR-B, marked in red, is ranked 25 of the 200 enriched genes.