The Oligonucleotide Binding (OB)-Fold Domain of KREPA4 Is Essential for Stable Incorporation into Editosomes

Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA) and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB)-fold proteins (KREPA1-A6), are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.     

The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei

Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by ∼20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C- terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3′-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates.     

The KREPA3 zinc finger motifs and OB-fold domain are essential for RNA editing and survival of Trypanosoma brucei

Three types of editosomes, each with an identical core containing six related KREPA proteins, catalyze the U insertion and deletion RNA editing of mitochondrial mRNAs in trypanosomes. Repression of expression of one of these, KREPA3 (also known as TbMP42), shows that it is essential for growth and in vivo editing in both procyclic (PF) and bloodstream (BF) life cycle stages of Trypanosoma brucei. RNA interference knockdown results in editosome disruption and altered in vitro editing in PFs, while repression by regulatable double knockout results in almost complete loss of editosomes in BFs. Mutational analysis shows that the KREPA3 zinc fingers and OB-fold domain are each essential for growth and in vivo editing. Nevertheless, KREPA3 with mutated zinc fingers incorporates into editosomes that catalyze in vitro editing and thus is not essential for editosome integrity, although stability is affected. In contrast, the OB-fold domain is essential for editosome integrity. Overall, KREPA3, especially its OB-fold, functions in editosome integrity, and its zinc fingers are essential for editing in vivo but not for the central catalytic steps. KREPA3 may function in editosome organization and/or RNA positioning.     

Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3′-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages.     

Endonuclease associations with three distinct editosomes in Trypanosoma brucei

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.     

Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodies

Several major global diseases are caused by single-cell parasites called trypanosomatids. These organisms exhibit many unusual features including a unique and essential U-insertion/deletion RNA editing process in their single mitochondrion. Many key RNA editing steps occur in ～20S editosomes, which have a core of 12 proteins. Among these, the "interaction protein" KREPA6 performs a central role in maintaining the integrity of the editosome core and also binds to ssRNA. The use of llama single domain antibodies (VHH domains) accelerated crystal growth of KREPA6 from Trypanosoma brucei dramatically. All three structures obtained are heterotetramers with a KREPA6 dimer in the center, and one VHH domain bound to each KREPA6 subunit. Two of the resultant heterotetramers use complementarity determining region 2 (CDR2) and framework residues to form a parallel pair of beta strands with KREPA6 - a mode of interaction not seen before in VHH domain-protein antigen complexes. The third type of VHH domain binds in a totally different manner to KREPA6. Intriguingly, while KREPA6 forms tetramers in solution adding either one of the three VHH domains results in the formation of a heterotetramer in solution, in perfect agreement with the crystal structures. Biochemical solution studies indicate that the C-terminal tail of KREPA6 is involved in the dimerization of KREPA6 dimers to form tetramers. The implications of these crystallographic and solution studies for possible modes of interaction of KREPA6 with its many binding partners in the editosome are discussed.     

A deletion site editing endonuclease in Trypanosoma brucei

RNA editing in Trypanosoma brucei inserts and deletes uridines in mitochondrial mRNAs by a series of enzymatic steps that are catalyzed by a multiprotein complex, the editosome. KREPB1 and two related editosome proteins KREPB2 and KREPB3 contain motifs that suggest endonuclease and RNA/protein interaction functions. Repression of KREPB1 expression in procyclic forms by RNAi inhibited growth, in vivo editing, and in vitro endoribonucleolytic cleavage of deletion substrates. However, cleavage of insertion substrates and the exoUase, TUTase, and ligase catalytic activities of editing were retained by 20S editosomes. Repression of expression of an ectopic KREPB1 allele in bloodstream forms lacking both endogenous alleles or exclusive expression of KREPB1 with point mutations in the putative RNase III catalytic domain also blocked growth, in vivo editing, and abolished cleavage of deletion substrates, without affecting the other editing steps. These data indicate that KREPB1 is an endoribonuclease that is specific for RNA editing deletion sites.     

Compositionally and functionally distinct editosomes in Trypanosoma brucei

Uridylate insertion/deletion RNA editing in Trypanosoma brucei mitochondria is catalyzed by a multiprotein complex, the approximately 20S editosome. Editosomes purified via three related tagged RNase III proteins, KREN1 (KREPB1/TbMP90), KREPB2 (TbMP67), and KREN2 (KREPB3/TbMP61), had very similar but nonidentical protein compositions, and only the tagged member of these three RNase III proteins was identified in each respective complex. Three new editosome proteins were also identified in these complexes. Each tagged complex catalyzed both precleaved insertion and deletion editing in vitro. However, KREN1 complexes cleaved deletion but not insertion editing sites in vitro, and, conversely, KREN2 complexes cleaved insertion but not deletion editing sites. These specific nuclease activities were abolished by mutations in the putative RNase III catalytic domain of the respective proteins. Thus editosomes appear to be heterogeneous in composition with KREN1 complexes catalyzing cleavage of deletion sites and KREN2 complexes cleaving insertion sites while both can catalyze the U addition, U removal, and ligation steps of editing.     

KREPB6, KREPB7, and KREPB8 are important for editing endonuclease function in Trypanosoma brucei

Three distinct editosomes are required for the uridine insertion/deletion editing that creates translatable mitochondrial mRNAs in Trypanosoma brucei. They contain KREPB6, KREPB7, or KREPB8 proteins and their respective endonucleases KREN3, KREN2, or KREN1. RNAi knockdowns of KREPB6, KREPB7, and KREPB8 variably affect growth and RNA editing. KREPB6 and KREPB7 knockdowns substantially reduced in vitro insertion site cleavage activity of their respective editosomes, while KREPB8 knockdown did not affect its editosome deletion site cleavage activity despite inhibition of growth and editing. KREPB6, KREPB7, and KREPB8 knockdowns disrupted tagged KREN3, KREN2, or KREN1 editosomes, respectively, to varying degrees, and in the case of KREN1 editosomes, the deletion editing site cleavage activity shifted to a smaller S value. The varying effects correlate with a combination of the relative abundances of the KREPB6-8 proteins and of the different insertion and deletion sites. Tagged KREPB6-8 were physically associated with deletion subcomplexes upon knockdown of the centrally interactive KREPA3 protein, while KREN1-3 endonucleases were associated with insertion subcomplexes. The results indicate that KREPB6-8 occupy similar positions in editosomes and are important for the activity and specificity of their respective endonucleases. This suggests that they contribute to the accurate recognition of the numerous similar but diverse editing site substrates.     

Differential functions of two editosome exoUases in Trypanosoma brucei

Mitochondrial RNAs in trypanosomes are edited by the insertion and deletion of uridine (U) nucleotides to form translatable mRNAs. Editing is catalyzed by three distinct editosomes that contain two related U-specific exonucleases (exoUases), KREX1 and KREX2, with the former present exclusively in KREN1 editosomes and the latter present in all editosomes. We show here that repression of KREX1 expression leads to a concomitant reduction of KREN1 in ∼20S editosomes, whereas KREX2 repression results in reductions of KREPA2 and KREL1 in ∼20S editosomes. Knockdown of KREX1 results in reduced cell viability, reduction of some edited RNA in vivo, and a significant reduction in deletion but not insertion endonuclease activity in vitro. In contrast, KREX2 knockdown does not affect cell growth or editing in vivo but results in modest reductions of both insertion and deletion endonuclease activities and a significant reduction of U removal in vitro. Simultaneous knockdown of both proteins leads to a more severe inhibition of cell growth and editing in vivo and an additive effect on endonuclease cleavage in vitro. Taken together, these results indicate that both KREX1 and KREX2 are important for retention of other proteins in editosomes, and suggest that the reduction in cell viability upon KREX1 knockdown is likely a consequence of KREN1 loss. Furthermore, although KREX2 appears dispensable for cell growth, the increased inhibition of editing and parasite viability upon knockdown of both KREX1 and KREX2 together suggests that both proteins have roles in editing.     

OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity

KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3′ oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem–loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.     

TbMP44 is essential for RNA editing and structural integrity of the editosome in Trypanosoma brucei

RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation of TbMP44 gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.     

Identification of novel components of Trypanosoma brucei editosomes

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed.     

RNA editing in Trypanosoma brucei requires three different editosomes

Trypanosoma brucei has three distinct ~20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ~20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.     

RNA editing: complexity and complications

RNA editing in Trypanosomatids creates functional mitochondrial mRNAs by extensive uridylate (U) insertion and deletion as specified by small guide RNAs (gRNAs). Editing is catalysed by the multiprotein editosome. Over 20 of its protein components have been identified and additional proteins are likely to function in editing and its regulation. The functions of only a few editosome proteins have been determined. Surprisingly, there are related pairs or sets of editosome proteins, and insertion and deletion editing appear to be functionally and perhaps spatially separate. A model for the editosome is proposed, which has a catalysis domain with separate sectors for insertion and deletion editing. It also contains domains for anchor duplex and upstream RNA binding, which position the sequence to be edited in the catalysis domain.     

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins. The pathway for mRNA/gRNA complex formation and assembly with the editosome is still unknown. Work from our laboratory has suggested that distinct mRNA/gRNA complexes anneal to form a conserved core structure that may be important for editosome assembly. The secondary structure for the apocytochrome b (CYb) pair has been previously determined and is consistant with our model of a three-helical structure. Here, we used cross-linking and solution structure probing experiments to determine the structure of the ATPase subunit 6 (A6) mRNA hybridized to its cognate gA6-14 gRNA in different stages of editing. Our results indicate that both unedited and partially edited A6/gA6-14 pairs fold into a three-helical structure similar to the previously characterized CYb/gCYb-558 pair. These results lead us to conclude that at least two mRNA/gRNA pairs with distinct editing sites and distinct primary sequences fold to a three-helical secondary configuration that persists through the first few editing events.