Genetic approaches to studying adenosine-to-inosine RNA editing

Increasing proteomic diversity via the hydrolytic deamination of adenosine to inosine (A-to-I) in select mRNA templates appears crucial to the correct functioning of the nervous system in several model organisms, including Drosophila, Caenorabditis elegans, and mice. The genome of the fruitfly, Drosophila melanogaster, contains a single gene encoding the enzyme responsible for deamination, termed ADAR (for adenosine deaminase acting on RNA). The mRNAs that form the substrates for ADAR primarily function in neuronal signaling, and, correspondingly, deletion of ADAR leads to severe nervous system defects. While several ADAR enzymes are present in mice, the presence of a single ADAR in Drosophila, combined with the diverse genetic toolkit available to researchers and the wide range of ADAR target mRNAs identified to date, make Drosophila an ideal organism to study the genetic basis of A-to-I RNA editing. This chapter describes a variety of methods for genetically manipulating Drosophila A-to-I editing both in time and space, as well as techniques to study the molecular basis of ADAR-mRNA interactions. A prerequisite for experiments in this field is the ability to quantify the levels of editing in a given mRNA. Therefore, several commonly used methods for the quantification of editing levels will also be described.