RESUMO
Identifying the PPR-E+-NUWA-DYW2 editosome improves our understanding of the C-to-U RNA editing in plant organelles. However, the mechanism of RNA editing remains to be elucidated. Here, we report that GLUTAMINE-RICH PROTEIN23 (GRP23), a previously identified nuclear transcription regulator, plays an essential role in mitochondrial RNA editing through interacting with MORF (multiple organellar RNA-editing factor) proteins and atypical DYW-type pentatricopeptide repeat (PPR) proteins. GRP23 is targeted to mitochondria, plastids, and nuclei. Analysis of the grp23 mutants rescued by embryo-specific complementation shows decreased editing efficiency at 352 sites in mitochondria and 6 sites in plastids, with a predominant specificity for sites edited by the PPR-E and PPR-DYW proteins. GRP23 interacts with atypical PPR-DYW proteins (MEF8, MEF8S, DYW2, and DYW4) and MORF proteins (MORF1 and MORF8), whereas the four PPR-DYWs interact with the two MORFs. These interactions may increase the stability of the GRP23-MORF-atypical PPR-DYW complex. Furthermore, analysis of mef8Nâ³64aamef8s double mutants shows that MEF8/MEF8S are required for the editing of the PPR-E protein-targeted sites in mitochondria. GRP23 could enhance the interaction between PPR-E and MEF8/MEF8S and form a homodimer or heterodimer with NUWA. Genetic complementation analysis shows that the C-terminal domains of GRP23 and NUWA possess a similar function, probably in the interaction with the MORFs. NUWA also interacts with atypical PPR-DYWs in yeast. Both GRP23 and NUWA interact with the atypical PPR-DYWs, suggesting that the PPR-E proteins recruit MEF8/MEF8S, whereas the PPR-E+ proteins specifically recruit DYW2 as the trans deaminase, and then GRP23, NUWA, and MORFs facilitate and/or stabilize the E or E+-type editosome formation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Edição de RNA , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Edição de RNA/genética , RNA Mitocondrial/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi-protein complex called the 'editosome' that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA-recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82-kDa protein with distinct domains, including a pair of Znf domains and a unique C-terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein-protein interaction assays that identified the C-terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site-specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA-binding domain. These functional characterizations of the Znfs and novel C-terminal domain contribute to our understanding of the model for the chloroplast plant editosome.
Assuntos
Arabidopsis/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Edição de RNA , RNA de Cloroplastos/genética , RNA de Plantas/genética , Dedos de Zinco/genéticaRESUMO
The genomes in the two energy-converting organelles of plant cells, chloroplasts and mitochondria, contain numerous 'errors' that are corrected at the level of RNA transcript copies. The genes encoded in the two endosymbiotic organelles would not function properly if their transcripts were not altered by site-specific cytidine-to-uridine (C-to-U) exchanges and by additional reverse U-to-C exchanges in hornworts, lycophytes, and ferns. These peculiar processes of plant RNA editing, re-establishing genetic information that could alternatively be present at the organelle genome level, has spurred much research over >30 years. Lately new studies have revealed numerous interesting insights, notably on the biochemical machinery identifying specific pyrimidine nucleobases for conversion from C to U and vice versa. Here, I will summarize prominent research findings that lately have contributed to our better understanding of these phenomena introducing an added layer of information processing in plant cells. Some of this recent progress is based on the successful functional expression of plant RNA editing factors in bacteria and mammalian cells. These research approaches have recapitulated natural processes of horizontal gene transfer through which some protist lineages seem to have acquired plant RNA editing factors and adapted them functionally for their own purposes.
Assuntos
Organelas , Edição de RNA , Uridina/genética , Uridina/metabolismo , Organelas/genética , Organelas/metabolismo , Plantas/genética , Plantas/metabolismo , Cloroplastos/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas de Plantas/metabolismoRESUMO
RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-ß nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix-turn-helix (HTH) superfamily of proteins with an α1α2α3ß1ß2 topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands.
Assuntos
DNA Forma Z , Trypanosoma brucei brucei , DNA Forma Z/metabolismo , RNA/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Edição de RNA , Citoplasma/metabolismo , Proteínas de Protozoários/químicaRESUMO
Trypanosome U-insertion/deletion RNA editing in mitochondrial mRNAs involves guide RNAs (gRNAs) and the auxiliary RNA editing substrate binding complex (RESC) and RNA editing helicase 2 complex (REH2C). RESC and REH2C stably copurify with editing mRNAs but the functional interplay between these complexes remains unclear. Most steady-state mRNAs are partially edited and include misedited "junction" regions that match neither pre-mRNA nor fully edited transcripts. Editing specificity is central to mitochondrial RNA maturation and function, but its basic control mechanisms remain unclear. Here we applied a novel nucleotide-resolution RNA-seq approach to examine ribosomal protein subunit 12 (RPS12) and ATPase subunit 6 (A6) mRNA transcripts. We directly compared transcripts associated with RESC and REH2C to those found in total mitochondrial RNA. RESC-associated transcripts exhibited site-preferential enrichments in total and accurate edits. REH2C loss-of-function induced similar substrate-specific and site-specific editing effects in total and RESC-associated RNA. It decreased total editing primarily at RPS12 5' positions but increased total editing at examined A6 3' positions. REH2C loss-of-function caused site-preferential loss of accurate editing in both transcripts. However, changes in total or accurate edits did not necessarily involve common sites. A few 5' nucleotides of the initiating gRNA (gRNA-1) directed accurate editing in both transcripts. However, in RPS12, two conserved 3'-terminal adenines in gRNA-1 could direct a noncanonical 2U-insertion that causes major pausing in 3'-5' progression. In A6, a noncanonical sequence element that depends on REH2C in a region normally targeted by the 3' half of gRNA-1 may hinder early editing progression. Overall, we defined transcript-specific effects of REH2C loss.
Assuntos
Proteínas de Protozoários/metabolismo , Edição de RNA , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma/metabolismo , Animais , Proteínas de Protozoários/genética , RNA Guia de Cinetoplastídeos , RNA Mensageiro/genética , RNA Mitocondrial/genética , RNA de Protozoário/genética , RNA-Seq , Especificidade por Substrato , Trypanosoma/genética , Trypanosoma brucei brucei/genéticaRESUMO
Pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing in plant mitochondria and plastids. In this study, we characterized maize (Zea mays) kernel mutant defective kernel 53 (dek53), which has an embryo lethal and collapsed endosperm phenotype. Dek53 encodes an E-subgroup PPR protein, which possesses a short PLS repeat region of only seven repeats. Subcellular localization analysis indicated that DEK53 is localized in the mitochondrion. Strand- and transcript-specific RNA-seq analysis showed that the dek53 mutation affected C-to-U RNA editing at more than 60 mitochondrial C targets. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the assembly of mitochondrial complex III in dek53. Transmission electron microscopic examination showed severe morphological defects of mitochondria in dek53 endosperm cells. In addition, yeast two-hybrid and luciferase complementation imaging assays indicated that DEK53 can interact with the mitochondrion-targeted non-PPR RNA editing factor ZmMORF1, suggesting that DEK53 might be a functional component of the organellar RNA editosome.
Assuntos
Regulação da Expressão Gênica de Plantas , Zea mays , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mitocondrial , Sementes/genética , Sementes/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMO
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 â¼20S 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 â¼20S 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.
Assuntos
Proteínas de Protozoários/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/metabolismo , Endonucleases/metabolismo , Técnicas de Silenciamento de Genes , Genes de Protozoários , Modelos Biológicos , Mutação , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonuclease III/química , Ribonuclease III/genética , Ribonuclease III/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei Editing is catalyzed by three distinct â¼20S 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.
Assuntos
Proteínas de Protozoários/metabolismo , Edição de RNA , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Biologia Computacional/métodos , Endonucleases/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteoma , Proteômica/métodos , Proteínas de Protozoários/química , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos TestesRESUMO
The pentatricopeptide repeat-DYW protein AtECB2 affects plastid RNA editing at seven sites, including accD-794, accD-1568, ndhF-290, ndhG-50, petL-5, rpoA-200 and rpoC1-488. To understand the mechanism of its involvement in RNA editing, a transgenic line was constructed with AtECB2 fused to a 4xMYC tag that could complement the atecb2 phenotype. RNA immunoprecipitation analysis indicated that AtECB2 is associated with the transcripts of accD, ndhF, ndhG and petL. Co-immunoprecipitation and mass spectrometry experiments showed that multiple organelle RNA editing factor 2 (MORF2) and porphobilinogen deaminase HEMC are associated with AtECB2. Biochemical analysis showed that AtECB2 directly interacts with HEMC through its E domain, while HEMC interacts with MORF8/RIP1. Deletion analysis showed that the E domain is essential for RNA editing. The hemc-1 mutant showed an albino and seedling-lethal phenotype. Of the seven editing sites affected in atecb2, the editing of accD-794 and ndhF-290 was also reduced in hemc-1. RNA immunoprecipitation analysis suggested that HEMC is associated with the editing sites of ndhF transcripts. These results showed that both HEMC and multiple organellar RNA editing factor (MORF) proteins are associated with AtECB2 for RNA editing in plastids.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Cloroplastos/metabolismo , Hidroximetilbilano Sintase/metabolismo , Edição de RNA , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clorofila/biossíntese , Proteínas de Cloroplastos/genética , Hidroximetilbilano Sintase/genética , Fator de Crescimento Insulin-Like II , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Fragmentos de Peptídeos , Fenótipo , Plastídeos/metabolismo , Precursores de Proteínas , RNA de Cloroplastos/genética , Plântula/enzimologia , Plântula/genética , Deleção de SequênciaRESUMO
Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes ("editosomes") are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 ((H2)F1 and (H2)F2). (H2)F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and (H2)F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and (H2)F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway.
Assuntos
Proteínas de Protozoários/metabolismo , Edição de RNA , RNA Helicases/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Pareamento de Bases , Bovinos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , RNA Helicases/química , RNA Guia de Cinetoplastídeos/química , RNA Mensageiro/química , Ribonucleoproteínas/química , Trypanosoma brucei brucei/química , Tripanossomíase Bovina/microbiologiaRESUMO
Plants have an RNA editing mechanism that prevents deleterious organelle mutations from resulting in impaired proteins. A typical flowering plant modifies about 40 cytidines in chloroplast transcripts and many hundreds of cytidines in mitochondrial transcripts. The plant editosome, the molecular machinery responsible for this process, contains members of several protein families, including the organelle RNA recognition motif (ORRM)-containing family. ORRM1 and ORRM6 are chloroplast editing factors, while ORRM2, ORRM3, and ORRM4 are mitochondrial editing factors. Here we report the identification of organelle RRM protein 5 (ORRM5) as a mitochondrial editing factor with a unique mode of action. Unlike other ORRM editing factors, the absence of ORRM5 in orrm5 mutant plants results in an increase of the editing extent in 14% of the mitochondrial sites surveyed. The orrm5 mutant also exhibits a reduced splicing efficiency of the first nad5 intron and slower growth and delayed flowering time. ORRM5 contains an RNA recognition motif (RRM) and a glycine-rich domain at the C terminus. The RRM provides the editing activity of ORRM5 and is able to complement the splicing but not the morphological defects.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Edição de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/crescimento & desenvolvimento , Mutação , Plantas Geneticamente Modificadas , Motivo de Reconhecimento de RNA , RNA Mitocondrial , Proteínas de Ligação a RNA/genéticaRESUMO
RNA editing is a posttranscriptional process that covalently alters the sequence of RNA molecules and plays important biological roles in both animals and land plants. In flowering plants, RNA editing converts specific cytidine residues to uridine in both plastid and mitochondrial transcripts. Previous studies identified pentatricopeptide repeat (PPR) motif-containing proteins as site-specific recognition factors for cytidine targets in RNA sequences. However, the regulatory mechanism underlying RNA editing was largely unknown. Here, we report that protoporphyrinogen IX oxidase 1 (PPO1), an enzyme that catalyzes protoporphyrinogen IX into protoporphyrin IX in the tetrapyrrole biosynthetic pathway, plays an unexpected role in editing multiple sites of plastid RNA transcripts, most of which encode subunits of the NADH dehydrogenase-like complex (NDH), in the reference plant Arabidopsis thaliana. We identified multiple organellar RNA editing factors (MORFs), including MORF2, MORF8, and MORF9, that interact with PPO1. We found that two conserved motifs within the 22-aa region at the N terminus of PPO1 are essential for its interaction with MORFs, its RNA editing function, and subsequently, its effect on NDH activity. However, transgenic plants lacking key domains for the tetrapyrrole biosynthetic activity of PPO1 exhibit normal RNA editing. Furthermore, MORF2 and MORF9 interact with three PPRs or related proteins required for editing of ndhB and ndhD sites. These results reveal that the tetrapyrrole biosynthetic enzyme PPO1 is required for plastid RNA editing, acting as a regulator that promotes the stability of MORF proteins through physical interaction.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Plastídeos/enzimologia , Plastídeos/genética , Protoporfirinogênio Oxidase/metabolismo , Edição de RNA/genética , Tetrapirróis/biossíntese , Proteínas de Arabidopsis/genética , Sequência de Bases , Clorofila/biossíntese , Flavina-Adenina Dinucleotídeo/metabolismo , Dados de Sequência Molecular , NADH Desidrogenase/metabolismo , Fenótipo , Ligação Proteica , Protoporfirinogênio Oxidase/genética , Plântula/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
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 â¼20S 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.
Assuntos
Estágios do Ciclo de Vida , Proteínas de Protozoários/metabolismo , Edição de RNA , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sangue/parasitologia , Linhagem Celular , Resistência a Medicamentos/genética , Estágios do Ciclo de Vida/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Ribonuclease III/química , Ribonuclease III/metabolismo , Tetraciclina/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismoRESUMO
Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors. Graphic abstract: Characteristics of the fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE) assay.
RESUMO
Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8 MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and as a consequence generates a simplified RNA folding landscape that is critical for the progression of the editing reaction cycle. Here we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (double-stranded) versus structurally unconstrained (single-stranded) nucleotides. Electrophilic anhydrides such as 1-methyl-7-nitroisatoic anhydride are used as probing reagents, and the ribose 2'-modified nucleotides are mapped as abortive cDNA synthesis products. As a result, SHAPE allows the identification of all single-stranded and base-paired regions in a given RNA, and the data are used to compute experimentally derived RNA 2D structures. A side-by-side comparison of the RNA 2D folds in the pre- and post-chaperone states finally maps the chaperone-induced dynamic of the different pre-mRNAs with single-nucleotide resolution.
Assuntos
Chaperonas Moleculares/metabolismo , Técnicas de Sonda Molecular , Proteínas de Protozoários/metabolismo , Edição de RNA , Dobramento de RNA , RNA Mitocondrial/química , RNA de Protozoário/química , RNA Mitocondrial/metabolismo , RNA de Protozoário/metabolismo , Análise de Sequência de RNA/métodos , Trypanosoma brucei bruceiRESUMO
RNA editing in chloroplasts and mitochondria is performed by hypothetical editosomes. The MORF family proteins are essential components of these editosomes. In Arabidopsis, MORF2 and MORF9 are involved in the editing of most sites in chloroplasts. In this work, we performed immunoprecipitation and mass spectrometry assays of transgenic lines expressing MORF2-4xMYC and MORF9-4xMYC to identify interacting proteins. We found that MORF2 and MORF9 are present in the same complex. Blue-Native PAGE analysis of chloroplast protein complexes also revealed that both MORF2 and MORF9 are part of a complex of approximately 140 kDa, suggesting the existence of tight MORF2-MORF9 interaction in chloroplasts. The editing of ndhD-1 (ndhD-C2) site was reported to be blocked in both morf2 and morf9. RNA immunoprecipitation assays showed that MORF2 and MORF9 are tightly associated with the editing site of ndhD-1. However, in an RNA-EMSA assay MORF2 and MORF9 could not directly bind to transcripts harboring the editing site of ndhD-1. Taken together, these results indicate that the MORF2-MORF9 heterodimer is the core members of editosomes in chloroplasts, while they are not responsible for RNA editing site recognition.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Cloroplastos/genética , Proteínas Mitocondriais/fisiologia , Edição de RNA , Proteínas de Ligação a RNA/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
RNA editing is a type of post-transcriptional modification that includes nucleotide insertion/deletion or conversion. Different categories of RNA editing have been widely observed in distinct RNAs from divergent organisms. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria, playing important roles in various plant developmental processes, including organelle biogenesis, adaptation to environmental changes, and signal transduction. Numerous studies have demonstrated that a number of factors are involved in plant RNA editing, such as pentatricopeptide repeat (PPR) proteins, multiple organelle RNA editing factors (MORF, also known as RIP), organelle RNA recognition motif (ORRM) containing proteins, protoporphyrinogen IX oxidase 1 (PPO1) and organelle zinc finger 1 (OZ1). These factors play diverse roles in plant RNA editing due to their distinct characteristics. In this review, we discuss the functional roles of the individual editing factors and their associations in plant RNA editing.
Assuntos
Organelas/metabolismo , Proteínas de Plantas/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Cloroplastos/metabolismo , Variação Genética , Mitocôndrias/metabolismo , Proteínas de Plantas/química , Plastídeos/metabolismo , Domínios Proteicos , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/químicaRESUMO
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.
RESUMO
RNA editing causes massive remodeling of the mitochondrial mRNA transcriptome in trypanosomes and related kinetoplastid protozoa. This type of editing involves the specific insertion or deletion of uridylates (U) directed by small noncoding guide RNAs (gRNAs). Because U-insertion exceeds U-deletion by a factor of 10, editing increases the nascent mRNA size by up to 55%. In Trypanosoma brucei, the editing apparatus uses ~40 proteins and >1,200 gRNAs to create the functional open reading frame in 12 mRNAs. Thousands of sites are specifically recognized in the pre-edited mRNAs and a myriad of partially edited transcript intermediates accumulates in mitochondria. The control of editing is poorly understood, but past work suggests that it occurs during substrate recognition, the initiation and progression of editing, and during the life-cycle in different hosts. The growing understanding of the editing proteins offers clues about editing control. Most editing proteins reside in the "RNA-free" RNA editing core complex (RECC) and in the accessory RNA editing substrate complex (RESC) that contains gRNA. Two accessory RNA helicases are known, including one in the RNA editing helicase 2 complex (REH2C). Both the RESC and the REH2C associate with mRNA, providing a rationale for the assembly of mRNA or its mRNPs, RESC, and the RECC enzyme. Identified variants of the canonical editing complexes further complicate the model of RNA editing. We examine specific examples of complex variants, differential effects of editing proteins on the mRNAs within and between T. brucei life stages, and possible control points in RNA holo-editosomes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Assuntos
Edição de RNA , Trypanosoma/genética , Proteínas de Protozoários/genética , RNA Mensageiro/genética , Ribonucleoproteínas/genéticaRESUMO
Most mitochondrial messenger RNAs in trypanosomatid pathogens undergo a unique type of posttranscriptional modification involving insertion and/or deletion of uridylates. This process, RNA editing, is catalyzed by a multiprotein complex (~1.6 MDa), the editosome. Knockdown of core editosome proteins compromises mitochondrial function and, ultimately, parasite viability. Hence, because the editosome is restricted to trypanosomatids, it serves as a unique drug target in these pathogens. Currently, there is a lack of editosome inhibitors for antitrypanosomatid drug development or that could serve as unique tools for perturbing and characterizing editosome interactions or RNA editing reaction stages. Here, we screened a library of pharmacologically active compounds (LOPAC1280) using high-throughput screening to identify RNA editing inhibitors. We report that aurintricarboxylic acid, mitoxantrone, PPNDS, and NF449 are potent inhibitors of deletion RNA editing (IC50 range, 1-5 µM). However, none of these compounds could specifically inhibit the catalytic steps of RNA editing. Mitoxantrone blocked editing by inducing RNA-protein aggregates, whereas the other three compounds interfered with editosome-RNA interactions to varying extents. Furthermore, NF449, a suramin analogue, was effective at killing Trypanosoma brucei in vitro. Thus, new tools for editosome characterization and downstream RNA editing inhibitor have been identified.