RESUMO
Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3'-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3'-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.
Assuntos
Mycobacterium tuberculosis , RNA de Transferência , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimologia , Especificidade por Substrato , RNA de Transferência/metabolismo , RNA de Transferência/química , Escherichia coli/genética , Escherichia coli/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Modelos Moleculares , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Citidina/química , Citidina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/genéticaRESUMO
Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.
Assuntos
Insuficiência de Crescimento , RNA Nucleotidiltransferases , Animais , Humanos , Camundongos , Camundongos Knockout , Debilidade Muscular/genética , Músculos , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/genética , Peixe-ZebraRESUMO
The maturation of tRNA and its quality control is crucial for aminoacylation and protein synthesis. The CCA enzyme, also known as tRNA nucleotidyltransferase, catalyzes the addition or repair of CCA at the 3'-terminus of tRNAs to facilitate aminoacylation. Structural studies of CCA enzyme in complex with ATP and CTP suggested that adding CCA at the 3'-terminus of tRNAs is a sequential process [1-4]. However, there are many inconsistent results of CCA addition from the biochemical studies, which raise the ambiguity about the CCA enzyme specificity in vitro [5-7]. On the other hand, there are no effective methods for preparing the 3'-amino-tailed tRNA to provide a stable amide linkage, which is vital to make homogeneous samples for structural studies of stalling peptides to understand ribosome mediated gene regulation [7-11]. In this study, we examined the functional specificity of the Class II CCA enzyme from E. coli, and optimized the benchmark experimental conditions to prepare the 3'-NH2-tRNA using the CCA enzyme. Our results suggest that the CCA enzyme has a specific ability to catalyze the CCA addition/repair activity within the stoichiometric range of the reactants, and excess amounts of nucleotides lead to non-specific polymerization of the tRNA. Further, we developed an efficient method for synthesizing 3'-amino tRNA, which can facilitate stable aminoacyl/peptidyl-tRNA preparation.
Assuntos
Escherichia coli , RNA de Transferência , Escherichia coli/metabolismo , RNA de Transferência/metabolismo , RNA Nucleotidiltransferases/química , Nucleotídeos , Processamento Pós-Transcricional do RNA , Biossíntese de ProteínasRESUMO
Previous studies indicated that the P-body components, CGH-1 and EDC-3 may play a crucial role in the regulation of lifespan in Caenorhabditis elegans. Homo sapiens DDX6 or Saccharomyces cerevisiae Dhh1p (CGH-1 in C. elegans) could form complexes with EDC3 (Edc3p in yeast), respectively, which is significant for translation inhibition and mRNA decay. However, it is currently unclear how CGH-1 can be recognized by EDC-3 in C. elegans. Here, we provided structural and biochemical insights into the interaction between CGH-1 and EDC-3. Combined with homology modeling, mutation, and ITC assays, we uncovered an interface between CGH-1 RecA2 domain and EDC-3 FDF-FEK. Additionally, GST-pulldown and co-localization experiments confirmed the interaction between CGH-1 and EDC-3 in vitro and in vivo. We also analyzed PATR-1-binding interface on CGH-1 RecA2 by ITC assays. Moreover, we unveiled the similarity and differences of the binding mode between EDC-3 and CAR-1 or PATR-1. Taken together, these findings provide insights into the recognition of DEAD-box protein CGH-1 by EDC-3 FDF-FEK motif, suggesting important functional implications.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/embriologia , RNA Helicases/química , RNA Nucleotidiltransferases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Humanos , Ligação Proteica , RNA Helicases/genética , RNA Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
A protein-RNA complex containing the RNA helicase CGH-1 and a germline specific RNA-binding protein CAR-1 is involved in various aspects of function in C. elegans. However, the structural basis for the assembly of this protein complex remains unclear. Here, we elucidate the molecular basis of the recognition of CGH-1 by CAR-1. Additionally, we found that the ATPase activity of CGH-1 is stimulated by NTL-1a MIF4G domain in vitro. Furthermore, we determined the structures of the two RecA-like domains of CGH-1 by X-ray crystallography at resolutions of 1.85 and 2.40 Å, respectively. Structural and biochemical approaches revealed a bipartite interface between CGH-1 RecA2 and the FDF-TFG motif of CAR-1. NMR and structure-based mutations in CGH-1 RecA2 or CAR-1 attenuated or disrupted CGH-1 binding to CAR-1, assessed by ITC and GST-pulldown in vitro. These findings provide insights into a conserved mechanism in the recognition of CGH-1 by CAR-1. Together, our data provide the missing physical links in understanding the assembly and function of CGH-1 and CAR-1 in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Aminoácidos/química , Animais , Sequência Conservada , Cristalografia por Raios X , Isótopos de Nitrogênio , Domínios Proteicos , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Pre-mRNA processing and mRNA stability play direct roles in controlling protein abundance in a cell. Before the mRNA can be translated into a protein, the introns in the pre-mRNA transcripts need to be removed by splicing, such that exons can be ligated together and can code for a protein. In this process, the function of the RNA lariat debranching enzyme or Dbr1 provides a rate-limiting step in the intron turnover process and possibly regulating the production of translation competent mRNAs. Surprising new roles of Dbr1 are emerging in cellular metabolism which extends beyond intron turnover processes, ranging from splicing regulation to translational control. In this review, we highlight the importance of the Dbr1 enzyme, its structure and how anomalies in its function could relate to various human diseases.
Assuntos
RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Encefalite Viral/enzimologia , Encefalite Viral/genética , HIV/enzimologia , HIV/genética , Humanos , Íntrons , Neoplasias/enzimologia , Neoplasias/genética , RNA Nucleotidiltransferases/químicaRESUMO
The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that-when inserted into the human enzyme-confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 3'-end of the tRNA primer in the catalytic core, dramatically increases the enzyme's substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.
Assuntos
Mermithoidea/enzimologia , RNA Nucleotidiltransferases/metabolismo , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Biocatálise , Humanos , Cinética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Nucleotidiltransferases/química , RNA de Transferência/química , Especificidade por SubstratoRESUMO
Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3'-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3' CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.
Assuntos
Proteínas de Bactérias/genética , Citosina/metabolismo , RNA Nucleotidiltransferases/genética , RNA/genética , Aspergillus nidulans/genética , Proteínas de Bactérias/química , Sítios de Ligação/genética , Cristalografia por Raios X , Citosina/química , Modelos Moleculares , Poli A/química , Poli A/genética , RNA Nucleotidiltransferases/química , Especificidade por SubstratoRESUMO
Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.
Assuntos
Sistemas CRISPR-Cas/genética , RNA Nucleotidiltransferases/genética , RNA/genética , Química Click , Edição de Genes , Modelos Moleculares , RNA/química , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
3' uridylation is an essential modification associated with coding and noncoding RNA degradation in eukaryotes. In Arabidopsis, HESO1 was first identified as the major nucleotidyl transferase that uridylates most unmethylated miRNAs, and URT1 was later reported to play a redundant but important role in miRNA uridylation when HESO1 is absent. Two enzymes work sequentially and collaboratively to tail different forms of the same miRNAs in vivo. For mRNA, however, URT1 becomes the main enzyme to uridylate the majority of mRNA and repairs their deadenylated ends to restore the binding site for Poly(A) Binding Protein (PABP). HESO1, on the other hand, targets mostly the mRNAs with very short oligo(A) tails and fails in fulfilling the same task. To understand the structural basis these two functional homologues possess for their different substrate preferences and catalytic behaviors, we first determined the crystal structures of URT1 in the absence and presence of UTP. Our structures, together with functional assay and sequence analysis, indicated that URT1 has a conserved UTP-recognition mechanism analogue to the terminal uridylyl transferases from other species whereas HESO1 may evolve separately to recognize UTP in a different way. Moreover, URT1 N552 may be an important residue in interacting with 3' nucleotide of RNA substrate. The URT1 structure we determined represents the first structure of uridylyl transferase from plants, shedding light on the mechanisms of URT1/HESO1-dependent RNA metabolism.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , RNA Nucleotidiltransferases/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , RNA Nucleotidiltransferases/metabolismo , Uridina Trifosfato/metabolismoRESUMO
Lin28-dependent oligo-uridylylation of precursor let-7 (pre-let-7) by terminal uridylyltransferase 4/7 (TUT4/7) represses let-7 expression by blocking Dicer processing, and regulates cell differentiation and proliferation. The interaction between the Lin28:pre-let-7 complex and the N-terminal Lin28-interacting module (LIM) of TUT4/7 is required for pre-let-7 oligo-uridylylation by the C-terminal catalytic module (CM) of TUT4/7. Here, we report crystallographic and biochemical analyses of the LIM of human TUT4. The LIM consists of the N-terminal Cys2His2-type zinc finger (ZF) and the non-catalytic nucleotidyltransferase domain (nc-NTD). The ZF of LIM adopts a distinct structural domain, and its structure is homologous to those of double-stranded RNA binding zinc fingers. The interaction between the ZF and pre-let-7 stabilizes the Lin28:pre-let-7:TUT4 ternary complex, and enhances the oligo-uridylylation reaction by the CM. Thus, the ZF in LIM and the zinc-knuckle in the CM, which interacts with the oligo-uridylylated tail, together facilitate Lin28-dependent pre-let-7 oligo-uridylylation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Humanos , MicroRNAs/genética , Ligação Proteica , RNA Nucleotidiltransferases/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Correct synthesis and maintenance of functional tRNA 3'-CCA-ends is a crucial prerequisite for aminoacylation and must be achieved by the phylogenetically diverse group of tRNA nucleotidyltransferases. While numerous reports on the in vitro characterization exist, robust analysis under in vivo conditions is lacking. Here, we utilize Escherichia coli RNase T, a tRNA-processing enzyme responsible for the tRNA-CCA-end turnover, to generate an in vivo system for the evaluation of A-adding activity. Expression of RNase T results in a prominent growth phenotype that renders the presence of a CCA- or A-adding enzyme essential for cell survival in an E. coli Δcca background. The distinct growth fitness allows for both complementation and selection of enzyme variants in a natural environment. We demonstrate the potential of our system via detection of altered catalytic efficiency and temperature sensitivity. Furthermore, we select functional enzyme variants out of a sequence pool carrying a randomized codon for a highly conserved position essential for catalysis. The presented E. coli-based approach opens up a wide field of future studies including the investigation of tRNA nucleotidyltransferases from all domains of life and the biological relevance of in vitro data concerning their functionality and mode of operation.
Assuntos
Escherichia coli/genética , Exorribonucleases/genética , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/genética , Aminoacilação/genética , Escherichia coli/crescimento & desenvolvimento , Exorribonucleases/química , Cinética , Conformação de Ácido Nucleico , Nucleotidiltransferases/química , RNA Nucleotidiltransferases/química , Processamento Pós-Transcricional do RNA/genéticaRESUMO
A specific cytidine-cytidine-adenosine (CCA) sequence is required at the 3'-terminus of all functional tRNAs. This sequence is added during tRNA maturation or repair by tRNA nucleotidyltransferase enzymes. While most eukaryotes have a single enzyme responsible for CCA addition, some bacteria have separate CC- and A-adding activities. The fungus, Schizosaccharomyces pombe, has two genes (cca1 and cca2) that are thought, based on predicted amino acid sequences, to encode tRNA nucleotidyltransferases. Here, we show that both genes together are required to complement a Saccharomyces cerevisiae strain bearing a null mutation in the single gene encoding its tRNA nucleotidyltransferase. Using enzyme assays we show further that the purified S. pombe cca1 gene product specifically adds two cytidine residues to a tRNA substrate lacking this sequence while the cca2 gene product specifically adds the terminal adenosine residue thereby completing the CCA sequence. These data indicate that S. pombe represents the first eukaryote known to have separate CC- and A-adding activities for tRNA maturation and repair. In addition, we propose that a novel structural change in a tRNA nucleotidyltransferase is responsible for defining a CC-adding enzyme.
Assuntos
RNA Nucleotidiltransferases/metabolismo , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Sequência Conservada , Evolução Molecular , Viabilidade Microbiana , RNA Nucleotidiltransferases/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Schizosaccharomyces/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
Non-templated 3'-uridylation of RNAs has emerged as an important mechanism for regulating the processing, stability and biological function of eukaryotic transcripts. In Drosophila, oligouridine tailing by the terminal uridylyl transferase (TUTase) Tailor of numerous RNAs induces their degradation by the exonuclease Dis3L2, which serves functional roles in RNA surveillance and mirtron RNA biogenesis. Tailor preferentially uridylates RNAs terminating in guanosine or uridine nucleotides but the structural basis underpinning its RNA substrate selectivity is unknown. Here, we report crystal structures of Tailor bound to a donor substrate analog or mono- and oligouridylated RNA products. These structures reveal specific amino acid residues involved in donor and acceptor substrate recognition, and complementary biochemical assays confirm the critical role of an active site arginine in conferring selectivity toward 3'-guanosine terminated RNAs. Notably, conservation of these active site features suggests that other eukaryotic TUTases, including mammalian TUT4 and TUT7, might exhibit similar, hitherto unknown, substrate selectivity. Together, these studies provide critical insights into the specificity of 3'-uridylation in eukaryotic post-transcriptional gene regulation.
Assuntos
Proteínas de Drosophila/química , Nucleotidiltransferases/química , RNA Nucleotidiltransferases/química , Animais , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Modelos Moleculares , Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/metabolismo , Especificidade por SubstratoRESUMO
Terminal uridylyl transferase (TUTase) is one type of enzyme that modifies RNA molecules by facilitating the post-transcriptional addition of uridyl ribonucleotides to their 3' ends. Recent researches have reported that Drosophila TUTase, Tailor, exhibits an intrinsic preference for RNA substrates ending in 3'G, distinguishing it from any other known TUTases. Through this unique feature, Tailor plays a crucial role as the repressor in the biogenesis pathway of splicing-derived mirtron pre-miRNAs. Here we describe crystal structures of core catalytic domain of Tailor and its complexes with RNA stretches 5'-AGU-3' and 5'-AGUU-3'. We demonstrate that R327 and N347 are two key residues contributing cooperatively to Tailor's preference for 3'G, and R327 may play an extra role in facilitating the extension of polyuridylation chain. We also demonstrate that conformational stability of the exit of RNA-binding groove also contributes significantly to Tailor's activity. Overall, our work reveals useful insights to explain why Drosophila Tailor can preferentially select RNA substrates ending in 3'G and provides important values for further understanding the biological significances of biogenesis pathway of mirtron in flies.
Assuntos
Proteínas de Drosophila/genética , Drosophila/enzimologia , Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/química , RNA/biossíntese , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico/genética , Drosophila/genética , Proteínas de Drosophila/química , Guanina/química , MicroRNAs/genética , Nucleotidiltransferases/química , RNA/genética , RNA Nucleotidiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , Especificidade por SubstratoRESUMO
The determination of conditions for the reproducible growth of well diffracting crystals is a critical step in every biocrystallographic study. On the occasion of a new structural biology project, several advanced crystallogenesis approaches were tested in order to increase the success rate of crystallization. These methods included screening by microseed matrix screening, optimization by counter-diffusion and crystal detection by trace fluorescent labeling, and are easily accessible to any laboratory. Their combination proved to be particularly efficient in the case of the target, a 48â kDa CCA-adding enzyme from the psychrophilic bacterium Planococcus halocryophilus. A workflow summarizes the overall strategy, which led to the production of crystals that diffracted to better than 2â Å resolution and may be of general interest for a variety of applications.
Assuntos
Proteínas de Bactérias/química , Cristalização/métodos , Planococcus (Bactéria)/enzimologia , RNA Nucleotidiltransferases/química , Cristalografia por Raios X , Escherichia coli/genética , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Proteínas Recombinantes/genética , Fluxo de TrabalhoRESUMO
The RNA lariat debranching enzyme (Dbr1) has different functions in RNA metabolism, such as hydrolyzing the 2'-5' linkage in intron lariats, positively influencing Ty1 and HIV-1 retrotransposition, and modulating snRNP recycling during splicing reactions. It seems that Dbr1 is one of the major players in RNA turnover. It is remarkable that of all the studies carried out to date with Dbr1, to our knowledge, none of them have evaluated the expression profile of the endogenous Dbr1 gene. In this work, we describe, for the first time, that Entamoeba histolytica EhDbr1 mRNA has a very short half-life (less than 30 min) and encodes a very stable protein that is present until trophozoite cultures die. We also show that the EhDbr1 protein is present in the nuclear periphery on the cytoplasmic basal side, contrary to the localization of human Dbr1. Comparing these results with previous hypotheses and with results from different organisms suggests that Dbr1 gene expression is finely tuned and conserved across eukaryotes. Experiments describing the aspects of Dbr1 gene expression and Dbr1 mRNA turnover as well as other functions of the protein need to be performed. Particularly, a special emphasis is needed on the protozoan parasite E. histolytica, the causative agent of amoebiasis, since even though it is a unicellular organism, it is an intron-rich eukaryote whose intron lariats seem to be open to avoid intron lariat accumulation and to process them in non-coding RNAs that might be involved in its virulence.
Assuntos
Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Regulação da Expressão Gênica , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Núcleo Celular/enzimologia , Estabilidade Enzimática , Perfilação da Expressão Gênica , RNA Nucleotidiltransferases/química , Estabilidade de RNARESUMO
RNA-protein interactions permeate biology. Transcription, translation, and splicing all hinge on the recognition of structured RNA elements by RNA-binding proteins. Models of RNA-protein interactions are generally limited to short linear motifs and structures because of the vast sequence sampling required to access longer elements. Here, we develop an integrated approach that calculates global pairwise interaction scores from in vitro selection and high-throughput sequencing. We examine four RNA-binding proteins of phage, viral, and human origin. Our approach reveals regulatory motifs, discriminates between regulated and non-regulated RNAs within their native genomic context, and correctly predicts the consequence of mutational events on binding activity. We design binding elements that improve binding activity in cells and infer mutational pathways that reveal permissive versus disruptive evolutionary trajectories between regulated motifs. These coupling landscapes are broadly applicable for the discovery and characterization of protein-RNA recognition at single nucleotide resolution.
Assuntos
Produtos do Gene tat/química , RNA Nucleotidiltransferases/química , Proteínas de Ligação a RNA/química , RNA/química , Proteínas Virais Reguladoras e Acessórias/química , Sequência de Aminoácidos , Bacteriófago lambda/química , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Imunodeficiência Bovina/química , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , RNA/genética , RNA/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de RNA , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virologia , RNA/química , RNA/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Encefalopatias Metabólicas Congênitas/patologia , Tronco Encefálico/patologia , Encefalite Viral/genética , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Herpesvirus Humano 1 , Humanos , Interferons/metabolismo , Íntrons/genética , Masculino , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Linhagem , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Receptor 3 Toll-Like/metabolismo , Replicação ViralRESUMO
Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.