RESUMEN
Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration. Canonical editing by gRNAs that specify protein-encoding mRNA sequences occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression at a major early checkpoint in mRNA ND7, involving helicase KREH2-dependent opposite modulation of canonical and non-canonical 'terminator' gRNA utilization. Terminator-programmed editing derails canonical editing and installs proposed repressive structure in 30% of the ND7 transcriptome. BSF-to-PCF differentiation in vitro recreated this negative control. Remarkably, KREH2-RNAi knockdown relieved repression and increased editing progression by reverting canonical/terminator gRNA utilization. ND7 transcripts lacking early terminator-directed editing in PCF exhibited similar negative editing control along the mRNA sequence, suggesting global modulation of gRNA utilization fidelity. The terminator is a 'moonlighting' gRNA also associated with mRNA COX3 canonical editing, so the gRNA transcriptome seems multifunctional. Thus, KREH2 is the first identified repressor in developmental editing control. This and our prior work support a model whereby KREH2 activates or represses editing in a stage and substrate-specific manner. KREH2's novel dual role tunes mitochondrial gene expression in either direction during development.
RESUMEN
U-insertion/deletion (U-indel) RNA editing in trypanosome mitochondria is directed by guide RNAs (gRNAs). This editing may developmentally control respiration in bloodstream forms (BSF) and insect procyclic forms (PCF). Holo-editosomes include the accessory RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C), but the specific proteins controlling differential editing remain unknown. Also, RNA editing appears highly error prone because most U-indels do not match the canonical pattern. However, despite extensive non-canonical editing of unknown functions, accurate canonical editing is required for normal cell growth. In PCF, REH2C controls editing fidelity in RESC-bound mRNAs. Here, we report that KREH2, a REH2C-associated helicase, developmentally controls programmed non-canonical editing, including an abundant 3' element in ATPase subunit 6 (A6) mRNA. The 3' element sequence is directed by a proposed novel regulatory gRNA. In PCF, KREH2 RNAi-knockdown up-regulates the 3' element, which establishes a stable structure hindering element removal by canonical initiator-gRNA-directed editing. In BSF, KREH2-knockdown does not up-regulate the 3' element but reduces its high abundance. Thus, KREH2 differentially controls extensive non-canonical editing and associated RNA structure via a novel regulatory gRNA, potentially hijacking factors as a 'molecular sponge'. Furthermore, this gRNA is bifunctional, serving in canonical CR4 mRNA editing whilst installing a structural element in A6 mRNA.
Asunto(s)
Trypanosoma brucei brucei , Trypanosoma , ARN Mensajero/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma/genética , ARN/genética , ARN Protozoario/genética , ARN Protozoario/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Protozoarias/metabolismo , Edición de ARN , ARN Mensajero/metabolismo , ARN Mitocondrial/metabolismo , ARN Protozoario/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma/metabolismo , Animales , Proteínas Protozoarias/genética , ARN Guía de Kinetoplastida , ARN Mensajero/genética , ARN Mitocondrial/genética , ARN Protozoario/genética , RNA-Seq , Especificidad por Sustrato , Trypanosoma/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Bacterial proteases are of great pharmaceutical importance and have a key role in various biological processes and in life cycle of several pathogens. New technology used for rational protein engineering as well improved delivery options will expand the potential pharmaceutical applications of proteases. The catalytic proteases belong to metalloproteases (EC.3.4.24) that comprise thermo lysine. The metalloproteases and their homologs have many important biotechnological and therapeutic applications. In the present study, a novel protease gene nprB was isolated from a thermophilic bacterium Streptomyces thermovulgaris and bioinformatics analyses were performed. PCR amplification and sequencing of nprB gene indicated an open reading frame of 178 aa (20191.18 Dalton). Based on protein sequence homology as well as conserved motifs and PTF domain the protein is characterized as a thermo lysine-like protease and is a member of M4 family of metalloproteases. Different bioinformatics tools such as ProtParam, SOPMA, signalP4.1 and ProDom from the ExPAsy server were used for structural and functional analyses. A phylogram was also reconstructed to reveal evolutionary relationships of nprB with its various homologs. The provided data will serve as a background to further reveal pharmaceutical and biotechnological importance of this novel protease gene from S. thermovulgaris in future.
Asunto(s)
Proteínas Bacterianas/metabolismo , Metaloproteasas/farmacología , Streptomyces/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Biología Computacional , Estabilidad de Enzimas , Metaloproteasas/genética , Metaloproteasas/aislamiento & purificación , Modelos Moleculares , Filogenia , Conformación Proteica , Streptomyces/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas Protozoarias/metabolismo , Edición de ARN , ARN Helicasas/metabolismo , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , Emparejamiento Base , Bovinos , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , ARN Helicasas/química , ARN Guía de Kinetoplastida/química , ARN Mensajero/química , Ribonucleoproteínas/química , Trypanosoma brucei brucei/química , Tripanosomiasis Bovina/microbiologíaRESUMEN
Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.
Asunto(s)
Kinetoplastida , Edición de ARN , ARN Guía de Kinetoplastida , ARN Protozoario/metabolismo , ARN/metabolismo , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Células Cultivadas , Kinetoplastida/genética , Kinetoplastida/metabolismo , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN/química , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Mitocondrial , ARN Protozoario/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6-8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries.
RESUMEN
The mitochondrial genome of kinetoplastids, including species of Trypanosoma and Leishmania, is an unprecedented DNA structure of catenated maxicircles and minicircles. Maxicircles represent the typical mitochondrial genome encoding components of the respiratory complexes and ribosomes. However, most mRNA sequences are cryptic, and their maturation requires a unique U insertion/deletion RNA editing. Minicircles encode hundreds of small guide RNAs (gRNAs) that partially anneal with unedited mRNAs and direct the extensive editing. Trypanosoma brucei gRNAs and mRNAs are transcribed as polycistronic precursors, which undergo processing preceding editing; however, the relevant nucleases are unknown. We report the identification and functional characterization of a close homolog of editing endonucleases, mRPN1 (mitochondrial RNA precursor-processing endonuclease 1), which is involved in gRNA biogenesis. Recombinant mRPN1 is a dimeric dsRNA-dependent endonuclease that requires Mg(2+), a critical catalytic carboxylate, and generates 2-nucleotide 3' overhangs. The cleavage specificity of mRPN1 is reminiscent of bacterial RNase III and thus is fundamentally distinct from editing endonucleases, which target a single scissile bond just 5' of short duplexes. An inducible knockdown of mRPN1 in T. brucei results in loss of gRNA and accumulation of precursor transcripts (pre-gRNAs), consistent with a role of mRPN1 in processing. mRPN1 stably associates with three proteins previously identified in relatively large complexes that do not contain mRPN1, and have been linked with multiple aspects of mitochondrial RNA metabolism. One protein, TbRGG2, directly binds mRPN1 and is thought to modulate gRNA utilization by editing complexes. The proposed participation of mRPN1 in processing of polycistronic RNA and its specific protein interactions in gRNA expression are discussed.
Asunto(s)
ARN Guía de Kinetoplastida/biosíntesis , Ribonucleasa III/metabolismo , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Regulación de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Ribonucleasa III/química , Alineación de Secuencia , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3'-terminal maturation, RNA editing, turnover, and translation; however, these processes remain poorly studied. Core editing complexes and their U-insertion/deletion activities are relatively well characterized, and a battery of ancillary factors has recently emerged. This study characterized a novel DExH-box RNA helicase, termed here REH2 (RNA editing associated helicase 2), in unique ribonucleoprotein complexes that exhibit unwinding and guide RNA binding activities, both of which required a double-stranded RNA-binding domain (dsRBD) and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression.
Asunto(s)
Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Proteínas Protozoarias/metabolismo , ARN Helicasas/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma brucei brucei/enzimología , Secuencias de Aminoácidos/fisiología , Animales , Mitocondrias/genética , Proteínas Mitocondriales/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Protozoarias/genética , Edición de ARN/fisiología , ARN Helicasas/genética , Estabilidad del ARN/fisiología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Ribonucleoproteínas/genética , Ribosomas/genética , Ribosomas/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.
Asunto(s)
Aldehídos/química , Tratamiento Farmacológico de COVID-19 , Enfermedad de Chagas/tratamiento farmacológico , Inhibidores de Cisteína Proteinasa/uso terapéutico , SARS-CoV-2/enzimología , Trypanosoma cruzi/enzimología , Aldehídos/metabolismo , Aldehídos/farmacología , Catepsina L/antagonistas & inhibidores , Catepsina L/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/química , Diseño de Fármacos , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , SARS-CoV-2/efectos de los fármacos , Relación Estructura-Actividad , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi, is an important drug target for Chagas disease. We describe here a new series of reversible but time-dependent inhibitors of cruzain, composed of a dipeptide scaffold appended to vinyl heterocycles meant to provide replacements for the irreversible reactive "warheads" of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-(N-methyl)-pyridine groups conferred reversible, time-dependent inhibition of cruzain (Ki* = 0.1-0.4 µM). These cruzain inhibitors exhibited moderate to excellent selectivity versus human cathepsins B, L, and S and showed no apparent toxicity to human cells but were effective in cell cultures of Trypanosoma brucei brucei (EC50 = 1-15 µM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC50 = 5-8 µM). PVHIs represent a new class of cruzain inhibitors that could progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.
Asunto(s)
Inhibidores de Cisteína Proteinasa/farmacología , Peptidomiméticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/farmacología , Compuestos de Vinilo/farmacología , Animales , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/metabolismo , Diseño de Fármacos , Pruebas de Enzimas , Humanos , Cinética , Ratones , Simulación del Acoplamiento Molecular , Mioblastos Cardíacos/efectos de los fármacos , Peptidomiméticos/síntesis química , Peptidomiméticos/metabolismo , Unión Proteica , Proteínas Protozoarias/metabolismo , Piridinas/síntesis química , Piridinas/metabolismo , Piridinas/farmacología , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Pirimidinas/farmacología , Tripanocidas/síntesis química , Tripanocidas/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Compuestos de Vinilo/síntesis química , Compuestos de Vinilo/metabolismoRESUMEN
Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.
Asunto(s)
Edición de ARN/fisiología , ARN Mitocondrial/metabolismo , ARN Protozoario/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , ARN Mitocondrial/genética , ARN Protozoario/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Uridylate insertion/deletion RNA editing in Trypanosoma brucei is a complex system that is not found in humans, so there is interest in targeting this system for drug development. This system uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromolecular trans factors around mRNA including the RNA-free RNA editing core complex (RECC) and auxiliary ribonucleoprotein (RNP) complexes. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 (H2F1). Both of these proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes, H2F1 recruits REH2 to the editing apparatus, and H2F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editing trans factors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and that impact the assembly and interactions of REH2C. H2F1 upregulation stabilized REH2 in vivo. Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2F1 but not its interactions with other examined editing components. This result suggests that most, if not all, fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is a bona fide RNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.
Asunto(s)
Edición de ARN , ARN Helicasas/metabolismo , Trypanosoma brucei brucei/enzimología , Humanos , Mutación , ARN Helicasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcriptoma , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/parasitologíaRESUMEN
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.
Asunto(s)
Edición de ARN , Trypanosoma/genética , Proteínas Protozoarias/genética , ARN Mensajero/genética , Ribonucleoproteínas/genéticaRESUMEN
Multisubunit RNA editing complexes recognize thousands of pre-mRNA sites in the single mitochondrion of trypanosomes. Specific determinants at each editing site must trigger the complexes to catalyze a complete cycle of either uridylate insertion or deletion. While a wealth of information on the protein composition and catalytic activities of these complexes is currently available, the precise mechanisms that govern substrate recognition and editing site specificity remain unknown. This chapter describes basic assays to visualize direct photocrosslinking interactions between purified editing complexes and targeted deletion and insertion sites in model substrates for full-round editing. It also illustrates how variations of these assays can be applied to examine the specificity of the editing enzyme/substrate association, and to dissect structural or biochemical requirements of both the substrates and enzyme complex.
Asunto(s)
Edición de ARN , ARN Protozoario/genética , ARN/química , Trypanosoma brucei brucei/metabolismo , Animales , Secuencia de Bases , Bioquímica/métodos , Catálisis , Reactivos de Enlaces Cruzados/química , Eliminación de Gen , Datos de Secuencia Molecular , Unión Proteica , Precursores del ARN/genética , Especificidad por SustratoRESUMEN
Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.
Asunto(s)
Edición de ARN , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/metabolismo , Trypanosoma brucei brucei/genética , Adenosina Trifosfato/metabolismo , Animales , Ligasas de Carbono-Oxígeno/genética , Ligasas de Carbono-Oxígeno/metabolismo , Fraccionamiento Químico , Mitocondrias/química , Mitocondrias/genética , ARN Ligasa (ATP)/clasificación , Volumetría , Trypanosoma brucei brucei/enzimologíaRESUMEN
Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature.
Asunto(s)
Edición de ARN , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Trypanosoma brucei brucei/genética , Nucleótidos de Uracilo/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Secuencia de Bases , Citocromos b/genética , Citocromos b/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a approximately 25kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [(32)P] at the first editing site. The approximately 25kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5'-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.
Asunto(s)
Proteínas Protozoarias/genética , Edición de ARN/genética , Precursores del ARN/genética , ARN Protozoario/genética , Trypanosoma brucei brucei/genética , Adenosina Trifosfato/genética , Animales , Secuencia de Bases , Reactivos de Enlaces Cruzados , Regulación de la Expresión Génica/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Peso Molecular , Conformación de Ácido Nucleico , ARN/genética , ARN Guía de Kinetoplastida/genética , ARN Nuclear Heterogéneo/genética , ARN Mensajero/genética , ARN MitocondrialRESUMEN
Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicaseâ¢zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100-400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicaseâ¢zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans.
RESUMEN
Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3' to 5' direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a 'core' complex of proteins but differ in the composition of the 'variable' proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.