Isolation of RNA Binding Proteins Involved in Insertion/Deletion Editing

RNA editing is a collective term referring to a plethora of reactions that ultimately lead to changes in RNA nucleotide sequences apart from splicing, 5′ capping, or 3′ end processing. In the mitochondria of trypanosomatids, insertion and deletion of uridines must occur, often on a massive scale, in order to generate functional messenger RNAs. The current state of knowledge perceives the editing machinery as a dynamic system, in which heterogeneous protein complexes undergo multiple transient RNA-protein interactions in the course of gRNA processing, gRNA-mRNA recognition, and the cascade of nucleolytic and phosphoryl transfer reactions that ultimately change the mRNA sequence. Identification of RNA binding proteins that interact with the mitochondrial RNAs, core editing complex, or contribute to mRNA stability is of critical importance to our understanding of the editing process. This chapter describes purification and characterization of three RNA binding proteins from kinetoplastid mitochondria that have been genetically demonstrated to affect RNA editing.