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.     

KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei

The 20S editosome, a multiprotein complex, catalyzes the editing of most mitochondrial mRNAs in trypanosomatids by uridylate insertion and deletion. RNAi mediated inactivation of expression of KREPA4 (previously TbMP24), a component of the 20S editosome, in procyclic form Trypanosoma brucei resulted in inhibition of cell growth, loss of RNA editing, and disappearance of 20S editosomes. Levels of MRP1 and REAP-1 proteins, which may have roles in editing but are not editosome components, were unaffected. Tagged KREPA4 protein is incorporated into 20S editosomes in vivo with no preference for either insertion or deletion subcomplexes. Consistent with its S1-like motif, recombinant KREPA4 protein binds synthetic gRNA with a preference for the 3' oligo (U) tail. These data suggest that KREPA4 is an RNA binding protein that may be specific for the gRNA Utail and also is important for 20S editosome stability.     

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.     

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.     

A Protein-Protein Interaction Map of Trypanosome ��20S Editosomes

Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ∼20S on glycerol gradients. These ∼20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ∼20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease. Here we present a protein-protein interaction map that was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. One of these proteins, KREPA6, interacts with the OB-fold zinc finger protein in each subcomplex that interconnects their two catalytic proteins. Another OB-fold protein, KREPA3, appears to link to the putative endonuclease subcomplex. These results reveal a physical organization that underlies the coordination of the various catalytic and substrate binding activities within the ∼20S editosomes during the editing process.     

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.     

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.     

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.     

Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1

Ribosomal protein S1, consisting of six contiguous OB-folds, is the largest ribosomal protein and is essential for translation initiation in Escherichia coli. S1 is also one of the three essential host-derived subunits of Qβ replicase, together with EF-Tu and EF-Ts, for Qβ RNA replication in E. coli. We analyzed the crystal structure of Qβ replicase, consisting of the virus-encoded RNA-dependent RNA polymerase (β-subunit), EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qβ RNA replication. Structural and biochemical studies revealed that the two N-terminal OB-folds of S1 anchor S1 onto the β-subunit, and the third OB-fold is mobile and protrudes beyond the surface of the β-subunit. The third OB-fold mainly interacts with a specific RNA fragment derived from the internal region of Qβ RNA, and its RNA-binding ability is required for replication initiation of Qβ RNA. Thus, the third mobile OB-fold of S1, which is spatially anchored near the surface of the β-subunit, primarily recruits the Qβ RNA toward the β-subunit, leading to the specific and efficient replication initiation of Qβ RNA, and S1 functions as a replication initiation factor, beyond its established function in protein synthesis.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.     

RNA sequence and base pairing effects on insertion editing in Trypanosoma brucei

RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.     

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 in Trypanosoma brucei: characterization of gRNA U-tail interactions with partially edited mRNA substrates

Guide RNAs (gRNAs), key components of the RNA editing reaction in Trypanosoma brucei, direct the insertion and deletion of uridylate (U) residues. Analyses of gRNAs reveal three functional elements. The 5′-end of the gRNA contains the anchor, which is responsible for selection and binding to the pre-edited mRNA. The second element (the guiding region) provides the information required for editing. At the 3′-end of the gRNA is a non-encoded U-tail, whose function remains unclear. However, the cleavage–ligation model for editing proposes that the U-tail binds to purine-rich regions upstream of editing sites, thereby strengthening the interaction and holding onto the 5′ cleavage product. Our previous studies demonstrated that the U-tail interacts with upstream sequences and may play roles in both stabilization and tethering. These studies also indicated that the U-tail interactions involved mRNA regions that were to be subsequently edited. This raised the question of what happens to the mRNA–U-tail interaction as editing proceeds in the 3′→5′ direction. We examined gCYb-558 and its U-tail interaction with 5′CYbUT and two partially edited 5′CYb substrates. Our results indicate that the 3′-end of the U-tail interacts with the same sequence in all three mRNAs. Predicted secondary structures using crosslinking data suggest that a similar structure is maintained as editing proceeds. These results indicate that the role of the U-tail may also involve maintenance of important secondary structure motifs.