The essential functions of KREPB4 are developmentally distinct and required for endonuclease association with editosomes.

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in , and several transcripts are differentially edited in bloodstream (BF) and procyclic form (PF) cells correlating with changes in mitochondrial function. Editing is catalyzed by three ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III KREN1, N2, and N3 endonucleases with distinct cleavage specificities. KREPB4 is a common editosome protein that has a degenerate RNase III domain lacking conserved catalytic residues, in addition to zinc-finger and Pumilio/fem-3 mRNA binding factor (PUF) motifs. Here we show that KREPB4 is essential for BF and PF growth, in vivo RNA editing, and editosome integrity, but that loss of KREPB4 has differential effects on editosome components and complexes between BF and PF cells. We used targeted mutagenesis to investigate the functions of the conserved PUF and RNase III domains in both life-cycle stages and show that the PUF motif is not essential for function in BF or PF. In contrast, specific mutations in the RNase III domain severely inhibit BF and PF growth and editing, and disrupt ∼20S editosomes, while others indicate that the RNase III domain is noncatalytic. We further show that KREPB4, specifically the noncatalytic RNase III domain, is required for the association of KREN1, N2, and N3 with PF editosomes. These results, combined with previous studies, support a model in which KREPB4 acts as a pseudoenzyme to form the noncatalytic half of an RNase III heterodimer with the editing endonucleases.