A method to find tissue-specific novel sites of selective adenosine deamination.
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.
Ohlson
Johan
J
Department of Molecular Biology and Functional Genomics, Stockholm University, S-106 91 Stockholm, Sweden.
Ensterö
Mats
M
Sjöberg
Britt-Marie
BM
Ohman
Marie
M
D000241:Adenosine / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D003641:Deamination; D047468:Immunoprecipitation; D007288:Inosine / Q000378:metabolism*; D051379:Mice; D023282:Nuclease Protection Assays; D020411:Oligonucleotide Array Sequence Analysis; D017393:RNA Editing; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016601:RNA-Binding Proteins; D007288:Inosine; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D000241:Adenosine