KRBP72 facilitates ATPase-dependent editing progression through a structural roadblock in mitochondrial A6 mRNA

Abstract

Uridine insertion/deletion editing of mitochondrial messenger RNAs (mRNAs) in kinetoplastids entails the coordinated action of three complexes. RNA Editing Catalytic Complexes (RECCs) catalyze the enzymatic reactions, while the RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C) coordinate interactions between RECCs, mRNAs and hundreds of guide RNAs that direct edited sequences. Additionally, numerous auxiliary factors are required for productive editing of specific mRNAs. Here, we elucidate the role of KRBP72, an editing auxiliary factor of the ABC adenosine triphosphatase (ATPase) family that exhibits RNA-binding activity. In procyclic form Trypanosoma brucei, KRBP72 knockdown leads to a pause in editing at the base of a predicted stem loop structure in adenosine triphosphate synthase subunit 6 (A6) mRNA. Enhanced cross-linking and affinity purification revealed KRBP72 binding sites both within and upstream of this stem loop. KRBP72 ATPase activity is essential for its A6 mRNA editing function; however, its RNA-binding activity is dispensable. KRBP72 interacts with most RESC proteins in an RNase-sensitive manner. By contrast, RESC12A associates with KRBP72 in an RNase-insensitive fashion, and RESC12A promotes KRBP72’s interaction with RNA. Hence, KRBP72 ATPase activity facilitates progression of editing through a challenging secondary structure, highlighting this protein's crucial role in A6 mRNA editing.