RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in .

Editosomes are the multiprotein complexes that catalyze the insertion and deletion of uridines to create translatable mRNAs in the mitochondria of kinetoplastids. Recognition and cleavage of a broad diversity of RNA substrates require three functionally distinct RNase III-type endonucleases， as well as five additional editosome proteins that contain noncatalytic RNase III domains. RNase III domains have recently been identified in the editosome accessory proteins KREPB9 and KREPB10， suggesting a role related to editing endonuclease function. In this report， we definitively show that KREPB9 and KREPB10 are not essential in either bloodstream-form parasites (BF) or procyclic-form parasites (PF) by creating null or conditional null cell lines. While preedited and edited transcripts are largely unaffected by the loss of KREPB9 in both PF and BF， loss of KREPB10 produces distinct responses in BF and PF. BF cells lacking KREPB10 also lack edited CYb， while PF cells have increased edited A6， RPS12， ND3， and COII after loss of KREPB10. We also demonstrate that mutation of the RNase III domain of either KREPB9 or KREPB10 results in decreased association with ~20S editosomes. Editosome interactions with KREPB9 and KREPB10 are therefore mediated by the noncatalytic RNase III domain， consistent with a role in endonuclease specialization in . is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in generates mature mitochondrial mRNAs. Editing is essential for survival in mammalian hosts and tsetse fly vectors and is differentially regulated during the parasite life cycle. Three multiprotein "editosomes，" typified by exclusive RNase III endonucleases that act at distinct sites， catalyze editing. Here， we show that editosome accessory proteins KREPB9 and KREPB10 are not essential for mammalian blood- or insect-form parasite survival but have specific and differential effects on edited RNA abundance in different stages. We also characterize KREPB9 and KREPB10 noncatalytic RNase III domains and show they are essential for editosome association， potentially via dimerization with RNase III domains in other editosome proteins. This work enhances the understanding of distinct editosome and accessory protein functions， and thus differential editing， during the parasite life cycle and highlights the importance of RNase III domain interactions to editosome architecture.