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

ABSTRACT: Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei, 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.

SUBMITTER: McDermott SM

PROVIDER: S-EPMC5648035 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

McDermott Suzanne M SM Stuart Kenneth K

RNA (New York, N.Y.) 20170811 11

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in <i>Trypanosoma brucei</i>, 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 p ...[more]

PMID: 28802260

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Project description: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 in vivo 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 Trypanosoma brucei. IMPORTANCETrypanosoma brucei is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in T. brucei 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.

Dataset Information

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

Publications

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

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