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Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

RNA editing is an essential post-transcriptional process that creates functional mitochondrial mRNAs in Kinetoplastids. Multiprotein editosomes catalyze pre-mRNA cleavage, uridine (U) insertion or deletion, and ligation as specified by guide RNAs. Three functionally and compositionally distinct editosomes differ by the mutually exclusive presence of the KREN1, KREN2 or KREN3 endonuclease and their associated partner proteins. Because endonuclease cleavage is a likely point of regulation for RNA editing, we elucidated endonuclease specificity in vivo. We used a mutant gamma ATP synthase allele (MGA) to circumvent the normal essentiality of the editing endonucleases, and created cell lines in which both alleles of one, two or all three of the endonucleases were deleted. Cells lacking multiple endonucleases had altered editosome sedimentation on glycerol gradients and substantial defects in overall editing. Deep sequencing analysis of RNAs from such cells revealed clear discrimination by editosomes between sites of deletion versus insertion editing and preferential but overlapping specificity for sites of insertion editing. Thus, endonuclease specificities in vivo are distinct but with some functional overlap. The overlapping specificities likely accommodate the more numerous sites of insertion versus deletion editing as editosomes collaborate to accurately edit thousands of distinct editing sites in vivo., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra- and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture., Competing Interests: The authors declare no conflict of interest.

The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. We show here that various point mutations to KREPB4 and KREPB5 identify essential domains, and suggest that these proteins do not themselves perform RNase III catalysis. The zinc finger of KREPB4 but not KREPB5 is essential for editosome integrity and parasite viability, and mutation of the RNase III signature motif in KREPB5 prevents integration into editosomes, which is lethal. Isolated TAP-tagged KREPB4 and KREPB5 complexes preferentially associate with components of the deletion subcomplex, providing additional insights into editosome architecture. A new alignment of editosome RNase III sequences from several kinetoplastid species implies that KREPB4 and KREPB5 lack catalytic activity and reveals that the PUF motif is present in the editing endonucleases KREN1, KREN2, and KREN3. The data presented here are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.

Three distinct editosomes, typified by mutually exclusive KREN1, KREN2, or KREN3 endonucleases, are essential for mitochondrial RNA editing in Trypanosoma brucei. The three editosomes differ in substrate endoribonucleolytic cleavage specificity, which may reflect the vast number of editing sites that need insertion or deletion of uridine nucleotides (Us). Each editosome requires the single RNase III domain in each endonuclease for catalysis. Studies reported here show that the editing endonucleases do not form homodimeric domains, and may therefore function as intermolecular heterodimers, perhaps with KREPB4 and/or KREPB5. Editosomes isolated via TAP tag fused to KREPB6, KREPB7, or KREPB8 have a common set of 12 proteins. In addition, KREN3 is only found in KREPB6 editosomes, KREN2 is only found in KREPB7 editosomes, and KREN1 is only found in KREPB8 editosomes. These are the same associations previously found in editosomes isolated via the TAP-tagged endonucleases KREN1, KREN2, or KREN3. Furthermore, TAP-tagged KREPB6, KREPB7, and KREPB8 complexes isolated from cells in which expression of their respective endonuclease were knocked down were disrupted and lacked the heterotrimeric insertion subcomplex (KRET2, KREPA1, and KREL2). These results and published data suggest that KREPB6, KREPB7, and KREPB8 associate with the deletion subcomplex, whereas the KREN1, KREN2, and KREN3 endonucleases associate with the insertion subcomplex.

Mitochondrial (mt) gene expression in Trypanosoma brucei entails multiple types of RNA processing, including polycistronic transcript cleavage, mRNA editing, gRNA oligouridylation, and mRNA polyadenylation, which are catalyzed by various multiprotein complexes. We examined the novel mitochondrial RNA-binding 1 (MRB1) complex that has 16 associated proteins, four of which have motifs suggesting RNA interaction. RNase treatment or the lack of kDNA in mutants resulted in lower MRB1 complex sedimentation in gradients, indicating that MRB1 complex associates with kDNA transcripts. RNAi knockdowns of expression of the Tb10.406.0050 (TbRGGm, RGG motif), Tb927.6.1680 (C2H2 zinc finger), and Tb11.02.5390 (no known motif) MRB1 proteins each inhibited in vitro growth of procyclic form parasites and resulted in cells with abnormal numbers of nuclei. Knockdown of TbRGGm, but not the other two proteins, disrupted the MRB1 complex, indicating that it, but perhaps not the other two, is required for complex assembly and/or stability. The knockdowns resulted in similar but nonidentical patterns of altered in vivo abundances of edited, pre-edited, and preprocessed mt mRNAs, but did not appreciably affect the abundances of mRNAs that do not get edited. These results indicate that MRB1 complex is critical to the processing of mt RNAs, and although its specific function is unknown, it appears essential to parasite viability.