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

Uridine insertion/deletion editing of mitochondrial 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 1 Complex coordinate interactions between RECCs, mRNAs, and hundreds of gRNAs 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 ATPase family that exhibits RNA binding activity. In procyclic form T. brucei, KRBP72 knockdown leads to a pause in editing at the base of a predicted stem loop structure in ATP 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 RNA-dependent manner. By contrast, RESC12A associates with KRBP72 in an RNA-independent fashion, and RESC12A strongly promotes KRBP72’s interaction with RNA. Hence, KRBP72 ATPase activity facilitates the progression of editing through a challenging RNA secondary structure, highlighting this protein’s crucial role in A6 mRNA editing. To investigate the function of KRBP72 in RNA editing, we generated a KRBP72 RNAi cell line. We then performed RNA-seq analysis on A6 mitochondrial transcripts in two biological replicates (rep1-doxy, rep2-doxy, rep1+doxy, and rep2+doxy). We analyzed these sequences using TREAT to identify the junction end sites in the KRBP72 knockdown cell line. To identify the direct binding sites of KRBP72, we generated a KRBP72-MHT cell line and performed eCLAP analysis in three biological replicates **Submitters' note: the RNAseq data was submitted to the Sequence Read Archive with accession number PRJNA1133815***