RNA Editing Complex Research Articles

Dissecting the molecular puzzle of the editosome core in Arabidopsis organelles

Over the last decade, the composition of the C-to-U RNA editing complex in embryophyte organelles has turned out to be much more complex than first expected. While PPR proteins were initially thought to act alone, significant evidences have clearly depicted a sophisticated mechanism with numerous protein-protein interaction involving PPR and non-PPR proteins. Moreover, the identification of specific functional partnership between PPRs also suggests that, in addition to the highly specific PPRs directly involved in the RNA target recognition, non-RNA-specific ones are required. Although some of them, such as DYW1 and DYW2, were shown to be the catalytic domains of the editing complex, the molecular function of others, such as NUWA, remains elusive. It was suggested that they might stabilize the complex by acting as a scaffold. We here performed functional complementation of the crr28–2 mutant with truncated CRR28 proteins mimicking PPR without the catalytic domain and show that they exhibit a specific dependency to one of the catalytic proteins DYW1 or DYW2. Moreover, we also characterized the role of the PPR NUWA in the editing reaction and show that it likely acts as a scaffolding factor. NUWA is no longer required for efficient editing of the CLB19 editing sites once this RNA specific PPR is fused to the DYW catalytic domain of its partner DYW2. Altogether, our results strongly support a flexible, evolutive and resilient editing complex in which RNA binding activity, editing activity and stabilization/scaffolding function can be provided by one or more PPRs.

OsTHA8 encodes a pentatricopeptide repeat protein required for RNA editing and splicing during rice chloroplast development

In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice (Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8 (thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat (PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths. The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor (OsMORF8) and thioredoxin z (OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice.

Structural basis for guide RNA selection by the RESC1-RESC2 complex.

Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of Trypanosoma brucei RESC1-RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5'-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5'-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs.

Finding the hot spot: identifying immune sensitive gastrointestinal tumors.

Although researchers have been trying to harness the immune system for over 100 years, the advent of immune checkpoint blockers (ICB) marks an era of significant clinical outcomes in various metastatic solid tumors, characterized by complete and durable responses. ICBs are monoclonal antibodies that target either of a pair of transmembrane molecules in tumors or T-cells involved in immune evasion. Currently 2 ICBs targeting the checkpoint program death 1 (PD-1), nivolumab and pembrolizumab, and one cytotoxic lymphocyte antigen-4 (CTLA-4) inhibitor (ipilimumab) are approved in gastrointestinal malignancies. We review herein the current evidence on predictive biomarkers for ICB response in gastrointestinal tumors. A review of literature based on the National Cancer Institute list of FDA-approved drugs for neoplasms and FDA-approved therapies at the FDA website was performed. An initial literature review was based on the American Association for Clinical Research meeting 2019, the American Society of Clinical Oncology meeting 2019 and the European Society of Medical Oncology 2019 proceedings. A systematic search of PubMed was performed involving MeSH browser terms such as biomarkers, immunotherapy, gastrointestinal diseases and neoplasms. When appropriate, American and British terms were used in the search. The most relevant predictor of response to ICBs is microsatellite instability (MSI) and the data is strongest for colorectal cancer. At least 3 prospective trials show evidence of PD-L1 as a predictive biomarker for ICB response in gastroesophageal malignancies. At least one prospective trial has described tumor mutational burden high (TMB-H), independent of MSI, as predictive of response in anal and biliary tract carcinomas. DNA Polymerase Epsilon (POLE) or delta (POL-D) mutations have been implicated in a subset of MSS colorectal cancer with TMB-H but this biomarker requires prospective validation. There is evolving data based on retrospective observations that gene alterations predicting acquired resistance and hyper-progression. Ongoing clinical research is assessing the role of the human microbiome and RNA-editing complex mutations as predictive biomarkers of response to ICBs. MSI has the strongest predictive power among current biomarkers for ICB response in gastrointestinal cancers. Data continue to accumulate from ongoing clinical trials and new biomarkers are emerging from pre-clinical studies, suggesting that drug combinations targeting pathways complimentary to the PD-1/PD-L1 axis inhibition will define a robust field of clinical research.

High APOBEC1 Complementation Factor Expression Positively Modulates the Proliferation, Invasion, and Migration of Endometrial Cancer Cells Through Regulating P53/P21 Signaling Pathway.

Background: APOBEC1 complementation factor (A1CF) is a component of the apolipoprotein-B messenger RNA editing complex that participates in various cellular processes and acts as an oncogene in many cancers. In this study, it was aimed to investigate the roles of A1CF and its potential mechanism in endometrial cancer (EC). Materials and Methods: Gene expression prolife was downloaded from The Cancer Genome Atlas database. Then Kaplan-Meier and Cox regression analyses were conducted to assess the prognostic value of A1CF in EC. Cell Counting Kit-8, plate clone formation, and transwell assays were used to estimate the functions of A1CF on the proliferation, invasion, and migration of EC cell. The gene set enrichment analysis was used to analyze the pathway that is enriched by A1CF, whereas quantitative real-time polymerase chain reaction and Western blot analyses were utilized to detect the mRNA and protein expression involved. Results: It was detected that the upregulated A1CF was enriched in P53/P21 signaling pathway and tightly associated with patients' age, stage, and death. Besides, high A1CF expression led to a shorter overall survival of patients and predicted a poor prognosis in EC. The overexpression of A1CF promoted the proliferation, invasion, and migration of EC cells, whereas the depletion of A1CF suppressed these processes. Moreover, P21 and P53 were reduced whereas cyclin D1 and proliferating cell nuclear antigen were induced along with the increasing of A1CF. However, the effects of silencing A1CF on these protein expressions were on the contrary. Conclusion: A1CF was highly expressed and closely related to the prognosis and progression of EC through the regulation of P53/P21 signaling pathway, providing a possible new therapy target site for EC.

What is the Functional Role of Organelle Zinc finger 1 in the Plant C‐to‐U RNA Editing Complex?

Higher plants utilize protein complexes to precisely edit hundreds of RNA transcripts in organelles. The mechanistic model of RNA editing involves a C‐to‐U deamination reaction facilitated through the interaction of the RNA with a protein complex. Complex models have been proposed, but the minimal “editosome” capable of catalyzing C‐to‐U changes and the functions of many of the protein components have not been revealed. This research focuses on describing the functional role of one critical protein component belonging to the RAN binding protein 2 type zinc fingers protein family, Organelle Zinc finger 1 (OZ1). Zinc finger proteins have been associated with RNA binding; though RNA binding and site recognition in editosomes is known to occur via PPR proteins, OZ1 may be interacting with a scaffold RNA sequence. Recombinant OZ1 from Arabidopsis thaliana was used to analyze zinc coordination and RNA binding of this zinc finger protein. Spectrophotometric analysis of OZ1 after immobilized metal affinity chromatography (IMAC) suggests nucleic acid association. ICP‐MS and ESI‐MS analysis of a truncated variant of OZ1, only the sequence containing its two zinc finger domains (OZ1zf), suggests that OZ1 is coordinating only one zinc ion instead of the proposed two. Computational analysis of OZ1 dependent sites in Brachypodium distachyon returned a potential motif conserved among OZ1‐dependent editing sites. Electrophoretic mobility shift assays (EMSAs) are used to test RNA binding and the motif. Understanding this protein will bring us a step closer to manipulating this precise editing machine for use in biotechnology and may be used for purposes of crop improvement.Support or Funding InformationFunding for this work was provided by NIH SCORE SC2 ID: SC2 GM122718 awarded to Dr. Michael L. Hayes and the NIH MBRS‐RISE R25 GM 061331‐17 grant in support of Alfredo Jimenez‐Salinas.

Novel Interactions of Adrenodoxin-Related [2Fe-2S] Plant Ferredoxins MFDX1 and MFDX2 Indicate Their Involvement in a Wide Spectrum of Functions in Plant Mitochondria

Abstract Electron transfer chains of plant organelles (both chloroplasts and mitochondria) contain their own special set of ferredoxins. The relatively recently described adrenodoxin-like [2Fe-2S]-ferredoxins MFDX1 and MFDX2 of plant mitochondria are among the least studied of these. Until now, the only established function for them is participation in the final stage of biotin biosynthesis. In this work, using genetic and biochemical approaches, we searched for possible partners of these proteins in the genomes and proteomes of tobacco (Nicotiana tabacum L.) and foxglove (Digitalis purpurea L.) plants. MORF9 protein, one of the auxiliary components of the RNA editing complex of organelles (editosome), was found among the most prominent protein partners of adrenodoxin-like [2Fe-2S] tobacco ferredoxins. According to the results obtained from the yeast two-hybrid system, NtMFDX1 and NtMFDX2 of tobacco also bind and interact productively with the previously uncharacterised long non-coding polyadenylated RNA, which, based on its structural features, is capable of regulating the function of a number of components of complexes I (Nad1, Nad5) and III (protein of the cytochrome c synthesis system CcmF) and contributes to the formation of Fe/S-clusters in the corresponding protein complexes of the respiratory chain of plant mitochondria. We found one of the main components of the thiazol synthase complex (mitochondrial protein DpTHI1) to be the partner of ferredoxin DpMFDX2 of Digitalis purpurea. Finally, additional arguments were obtained in favour of the possible participation of MFDX1 and MFDX2 in the very ancient, but only recently described ‘progesterone’ steroid hormonal regulatory system: in leaves of the previously constructed CYP11A1-transgenic tomato plants, only the mature form of mitochondrial cytochrome P450scc (CYP11A1) of mammals is able to enter the mitochondria, where the above-mentioned components of the electron transport chain are localised. In summary, all of the newly revealed interactions of adrenodoxin-like [2Fe-2S] ferredoxins MFDX1 and MFDX2 indicate their participation in a wide range of functions in plant mitochondria.

Analysis of Tissue-specific RNA Editing Events of Genes Involved in RNA Editing in Arabidopsis thaliana

In plants, RNA editing is a post-transcriptional modification of transcripts and commonly occurs in plastids and mitochondria. In the case of flowering plants, not only PPR but also non-PPR proteins, such as MORF/RIP, ORRM and OZ partake in the diverse RNA editing complex. In Arabidopsis thaliana, 12 types of RNA editing patterns have been predicted in nuclear, chloroplast, and mitochondrial transcripts. In this study, tissue-specific RNA editing events were detected in gene families involved in RNA editing. Different Arabidopsis tissues at variable developmental stages, including 4-, 8-, and 12-d-old seedlings and 16-, 21-, 27-, and 32-d-old leaves, stems, stipes and roots, were collected and used for this study. Nine types of RNA editing events, including C-to-U, U-to-C, A-to-I, A-to-C, A-to-U, G-to-A, G-to-C, U-to-A and U-to-G, were identified in target genes. Most of the editing events occurred in seedlings and leaves and a few in stem tissues. Extensive U-to-C editing (60%) and A-to-G editing (54%) was detected in 12-d-old seedlings and 21-d-old leaves, respectively. This is the first experimental report of tissue-specific nuclear RNA editing events found in plants. This study will provide important information for revealing the mechanism of RNA editing in plants.

SMK6 mediates the C-to-U editing at multiple sites in maize mitochondria

The recently identified PPR-E+/NVWA/DYW2 RNA editing complex provides insights into the mechanism of RNA editing in higher plant organelles. However, whether the complex works together with the previously identified editing factors RIPs/MORFs is unclear. In this paper, we identified a maize Smk6 gene, which encodes a mitochondrion-targeted PPR-E+protein with E1 and E2 domains at the C terminus. Loss of Smk6 function affects the C-to-U editing at nad1-740, nad4L-110, nad7-739, and mttB-138,139 sites, impairs mitochondrial activity and blocks embryogenesis and endosperm development. Genetic and molecular analysis indicated that SMK6 is the maize ortholog of the Arabidopsis SLO2, which is a component of the PPR-E+/NVWA/DYW2 editing complex. However, yeast two-hybrid analyses did not detect any interaction between SMK6 and any of the mitochondrion-targeted RIPs/MORFs, suggesting that RIPs/MORFs may not be a component of PPR-E+/NVWA/DYW2 RNA editing complex. Further analyses are required to provide evidence that RIP/MORFs and SMK6 do not physically interact in vivo.

The RNA binding activity of the first identified trypanosome protein with Z-DNA-binding domains

RNA-binding proteins play a particularly important role in regulating gene expression in trypanosomes. A map of the network of protein complexes in Trypanosoma brucei uncovered an essential protein (Tb927.10.7910) that is postulated to be an RNA-binding protein implicated in the regulation of the mitochondrial post-transcriptional gene regulatory network by its association with proteins that participate in a multi-protein RNA editing complex. However, the mechanism by which this protein interacts with its multiple target transcripts remained unknown. Using sensitive database searches and experimental data, we identify Z-DNA-binding domains in T. brucei in the N- and C-terminal regions of Tb927.10.7910. RNA-binding studies of the wild-type protein, now referred to as RBP7910 (RNA binding protein 7910), and site-directed mutagenesis of residues important for the Z-DNA binding domains show that it preferentially interacts with RNA molecules containing poly(U) and poly(AU)-rich sequences. The interaction of RBP7910 with these regions may be involved in regulation of RNA editing of mitochondrial transcripts.

Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue-specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse versus human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets.

RNA editing of plastid-encoded genes

RNA editing is post-transcriptional modification to RNA molecules. In plants, RNA editing primarily occurs to two energy-producing organelles: plastids and mitochondria. Organelle RNA editing is often viewed as a mechanism of correction to compensate for defects or mutations in haploid organelle genomes. A common type of organelle RNA editing is deamination from cytidine to uridine. Cytidine-to-uridine plastid RNA editing is carried out by the RNA editing complex which consists of at least four types of proteins: pentatricopeptide repeat proteins, RNA editing interacting proteins/multiple organellar RNA editing factors, organelle RNA recognition motif proteins, and organelle zinc-finger proteins. The four types of RNA editing factors work together to carry out RNA editing site recognition, zinc cofactor binding, and cytidine-to-uridine deamination. In addition, three other types of proteins have been found to be important for plastid RNA editing. These additional proteins may play a regulatory or stabilizing role in the RNA editing complex.

Apobec-1 complementation factor regulates cell migration and apoptosis through Dickkopf1 by acting on its 3' untranslated region in MCF7 cells.

A1CF (apobec-1 complementation factor) acts as a component of the apolipoprotein-B messenger RNA editing complex. Previous researches mainly focused on its post-transcriptional cytidine to uridine RNA editing. However, few study reported its role in progression of breast carcinoma cells. Wound healing assay and flow cytometry were applied to detect the migration and apoptosis; western blot, real-time polymerase chain reaction, and dual-luciferase assays were applied to investigate the potential regulation mechanism of A1CF-mediated cell migration and apoptosis. Knockdown of A1CF decreased cell migration and enhanced cell apoptosis in MCF7 cells in vitro. Western blot analysis showed that knockdown of A1CF decreased Dickkopf1 but increased c-Myc and β-catenin expression, and overexpression of A1CF can get opposite results. Knockdown of Dickkopf1 in A1CF-overexpressed cells decreased cell migration and enhanced cell apoptosis compared with A1CF-overexpressed cells. Luciferase-fused 3' untranslated region of human Dickkopf1 activity was highly upregulated in A1CF-overexpressed MCF7 cells, but this upregulation can be inhibited by mutating conserved binding motifs of Dickkopf1 3' untranslated region. A1CF played a crucial role in cell migration and survival through affecting 3' untranslated region of Dickkopf1 to upregulate its expression in MCF7 cells.

The conserved domain in MORF proteins has distinct affinities to the PPR and E elements in PPR RNA editing factors

In plant organelles specific nucleotide motifs at C to U RNA editing sites are recognized by the PLS-class of pentatricopeptide repeat (PPR) proteins, which are additionally characterized by a C-terminal E domain. The PPR elements bind the nucleotides in the target RNA, while the function of the E domain has remained unknown. At most sites RNA editing also requires multiple organellar RNA editing factor (MORF) proteins. To understand how these two types of proteins are involved in RNA editing complexes, we systematically analyzed their protein-protein interactions. In vivo pull-down and yeast two-hybrid assays show that MORF proteins connect with selected PPR proteins. In a loss of function mutant of MORF1, a single amino acid alteration in the conserved MORF domain abrogates interactions with many PLS-class PPR proteins, implying the requirement of direct interaction to PPR proteins for the RNA editing function of MORF1. Subfragment analyses show that predominantly the N-terminal/central regions of the MORF domain in MORF1 and MORF3 bind the PPR proteins. Within the PPR proteins, the E domains in addition to PPR elements contact MORF proteins. In chimeric PPR proteins, different E domains alter the specificity of the interaction with MORF proteins. The selective interactions between E domain containing PPR and MORF proteins suggest that the E domains and MORF proteins play a key role for specific protein complexes to assemble at different RNA editing sites.

Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing

ABSTRACTThe mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg300-to-Trp300 and Pro313-to-Ser313 amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles.

Kinetoplast DNA-encoded ribosomal protein S12

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.

The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2.

Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ∼20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1.

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.

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of approximately 20S and approximately 35-40S and present the three-dimensional structures of both complexes by electron microscopy. The approximately 35-40S complexes consist of a platform density packed against a semispherical element. The approximately 20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The approximately 20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.

Determinants for Association and Guide RNA-Directed Endonuclease Cleavage by Purified RNA Editing Complexes from Trypanosoma brucei

U-insertion/deletion RNA editing in the single mitochondrion of kinetoplastids, an ancient lineage of eukaryotes, is a unique mRNA maturation process needed for translation. Multisubunit editing complexes recognize many pre-edited mRNA sites and modify them via cycles of three catalytic steps: guide RNA (gRNA)-directed cleavage, insertion or deletion of uridylates at the 3′-terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. This study defined relatively simple single- and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with UV photocrosslinking and functional studies. The cleaved strand required a minimal 5′ overhang of 12 nt and an ∼15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports in the context of purified native editing complexes show that the determinants for association, cleavage and full-round editing gradually increase in complexity as these stages progress. The native complexes in these studies contained most, if not all, known core subunits in addition to components of the MRP complex. Finally, we found that the endonuclease KREN1 in purified complexes photocrosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.