RESUMO
TRIM25 is an RNA-binding ubiquitin E3 ligase with central but poorly understood roles in the innate immune response to RNA viruses. The link between TRIM25's RNA binding and its role in innate immunity has not been established. Thus, we utilized a multitude of biophysical techniques to identify key RNA-binding residues of TRIM25 and developed an RNA-binding deficient mutant (TRIM25-m9). Using iCLIP2 in virus-infected and uninfected cells, we identified TRIM25's RNA sequence and structure specificity, that it binds specifically to viral RNA, and that the interaction with RNA is critical for its antiviral activity.
Assuntos
Ligação Proteica , RNA Viral , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , RNA Viral/metabolismo , RNA Viral/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células HEK293 , Imunidade Inata , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Antivirais/metabolismo , Antivirais/farmacologia , Vírus de RNA/genética , Sítios de LigaçãoRESUMO
A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Espalhamento a Baixo Ângulo , Fatores de Transcrição/ultraestrutura , Difração de Raios XRESUMO
RNA-binding proteins (RBPs) commonly feature multiple RNA-binding domains (RBDs), which provide these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability define the specificity of their interactions with RNA. However, how multiple RBDs recognize their cognate single-stranded RNA (ssRNA) sequences in concert remains poorly understood. Here, we use Upstream of N-Ras (Unr) as a model system to address this question. Although reported to contain five ssRNA-binding cold-shock domains (CSDs), we demonstrate that Unr includes an additional four CSDs that do not bind RNA (pseudo-RBDs) but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Disrupting the interactions between canonical and non-canonical CSDs impacts RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies reveal a new paradigm in protein-RNA recognition, where interactions between RBDs and pseudo-RBDs select RNA tertiary structures, influence RNP assembly, and define target specificity.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Sequência de Aminoácidos , Animais , Drosophila melanogaster , Biossíntese de Proteínas , Domínios ProteicosRESUMO
Protein-protein and protein-ligand interactions often involve conformational changes or structural rearrangements that can be quantified by solution small-angle X-ray scattering (SAXS). These scattering intensity measurements reveal structural details of the bound complex, the number of species involved and, additionally, the strength of interactions if carried out as a titration. Although a core part of structural biology workflows, SAXS-based titrations are not commonly used in drug discovery contexts. This is because prior knowledge of expected sample requirements, throughput and prediction accuracy is needed to develop reliable ligand screens. This study presents the use of the histidine-binding protein (26â kDa) and other periplasmic binding proteins to benchmark ligand screen performance. Sample concentrations and exposure times were varied across multiple screening trials at four beamlines to investigate the accuracy and precision of affinity prediction. The volatility ratio between titrated scattering curves and a common apo reference is found to most reliably capture the extent of structural and population changes. This obviates the need to explicitly model scattering intensities of bound complexes, which can be strongly ligand-dependent. Where the dissociation constant is within 102 of the protein concentration and the total exposure times exceed 20â s, the titration protocol presented at 0.5â mg ml-1 yields affinities comparable to isothermal titration calorimetry measurements. Estimated throughput ranges between 20 and 100 ligand titrations per day at current synchrotron beamlines, with the limiting step imposed by sample handling and cleaning procedures.
RESUMO
Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.
Assuntos
Proteínas Cromossômicas não Histona/química , DNA Helicases/química , Proteínas de Drosophila/química , Drosophila melanogaster/química , RNA Longo não Codificante/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , DNA Helicases/genética , DNA Helicases/metabolismo , Mecanismo Genético de Compensação de Dose , Motivo de Ligação ao RNA de Cadeia Dupla , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Masculino , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Subsequent to the publication of the above article, it has been noticed that data published in Figure 2A and Figure 2B of this article duplicate images previously published by this research group in the following paper.
RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0043651.].
RESUMO
We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural information on biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pairwise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl). The screening of 35 RNA motifs between 4 to 10 nucleotides reveals a strong variation of resulting complexes, revealed to be preference-switching between 1:1 and 2:2 binding stoichiometries upon addition of structural modeling. Validation of select sequences in isothermal calorimetry and NMR titration retrieves domain-specific roles and function of a guanine anchor. These findings reinforce the suitability of SAXS as a complement in lead identification.
Assuntos
Proteínas de Ligação a RNA/química , RNA/química , Espalhamento a Baixo Ângulo , Sítios de Ligação , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Termodinâmica , Raios XRESUMO
RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.
Assuntos
Archaea/enzimologia , Enzimas/metabolismo , RNA Ribossômico/genética , Metilação , Mutação , Conformação de Ácido Nucleico , RNA Ribossômico/químicaRESUMO
The constitutive decay element (CDE) of tumor necrosis factor α (TNF-α) mRNA (Tnf) represents the prototype of a class of RNA motifs that mediate rapid degradation of mRNAs encoding regulators of the immune response and development. CDE-type RNAs are hairpin structures featuring a tri-nucleotide loop. The protein Roquin recognizes CDE-type stem loops and recruits the Ccr4-Caf1-Not deadenylase complex to the mRNA, thereby inducing its decay. Stem recognition does not involve nucleotide bases; however, there is a strong stem sequence requirement for functional CDEs. Here, we present the solution structures of the natural Tnf CDE and of a CDE mutant with impaired Roquin binding. We find that the two CDEs adopt unique and distinct structures in both the loop and the stem, which explains the ability of Roquin to recognize stem loops in a sequence-specific manner. Our findings result in a relaxed consensus motif for prediction of new CDE stem loops.
Assuntos
Estabilidade de RNA , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Ubiquitina-Proteína Ligases/química , Animais , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , RNA Mensageiro/química , Processamento Alternativo , Animais , Drosophila melanogaster/química , Éxons , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Oócitos/metabolismo , RNA Mensageiro/genéticaRESUMO
During infection of macrophages, Mycobacterium tuberculosis, the pathogen that causes tuberculosis, utilizes fatty acids as a major carbon source. However, little is known about the coordination of the central carbon metabolism of M. tuberculosis with its chromosomal replication, particularly during infection. A recently characterized transcription factor called PrpR is known to directly regulate the genes involved in fatty acid catabolism by M. tuberculosis. Here, we report for the first time that PrpR also regulates the dnaA gene, which encodes the DnaA initiator protein responsible for initiating chromosomal replication. Using cell-free systems and intact cells, we demonstrated an interaction between PrpR and the dnaA promoter region. Moreover, real-time quantitative reverse-transcription PCR analysis revealed that PrpR acts as a transcriptional repressor of dnaA when propionate (a product of odd-chain-length fatty acid catabolism) was used as the sole carbon source. We hypothesize that PrpR may be an important element of the complex regulatory system(s) required for tubercle bacilli to survive within macrophages, presumably coordinating the catabolism of host-derived fatty acids with chromosomal replication.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Propionatos/metabolismo , Proteínas Repressoras/metabolismo , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
RNA-catalyzed lariat formation is present in both eukaryotes and prokaryotes. To date we lack structural insights into the catalytic mechanism of lariat-forming ribozymes. Here, we study an artificial 2'-5' AG1 lariat-forming ribozyme that shares the sequence specificity of lariat formation with the pre-mRNA splicing reaction. Using NMR, we solve the structure of the inactive state of the ribozyme in the absence of magnesium. The reaction center 5'-guanosine appears to be part of a helix with an exceptionally widened major groove, while the lariat-forming A48 is looped out at the apex of a pseudoknot. The model of the active state built by mutational analysis, molecular modeling, and small-angle X-ray scattering suggests that A48 is recognized by a conserved adenosine, juxtaposed to the 5'-guanosine in one base-pair step distance, while the G1-N7 coordinates a magnesium ion essential for the activation of the nucleophile. Our findings offer implications for lariat formation in RNA enzymes including the mechanism of the recognition of the branch-site adenosine.
Assuntos
RNA Catalítico/química , Sequência de Bases , Sítios de Ligação , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA Catalítico/metabolismoRESUMO
Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of odd-chain-length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell-free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd-chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Citratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Hidroliases/genética , Hidroliases/metabolismo , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
AdpA is a key regulator of morphological differentiation in Streptomyces. In contrast to Streptomyces griseus, relatively little is known about AdpA protein functions in Streptomyces coelicolor. Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA (adpA(Sc)) gene; the level of S. coelicolor AdpA (AdpA(Sc)) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpA(Sc) specifically binds the adpA(Sc) promoter region in vitro and in vivo, suggesting that its expression is autoregulated; surprisingly, in contrast to S. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpA(Sc) on the expression of several genes whose products play key roles in the differentiation of S. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpA(Sc) protein on the expression of the analyzed genes presumably results mainly from different affinities of AdpA(Sc) protein to individual promoters.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismo , Transativadores/metabolismo , Proteínas de Bactérias/genética , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Streptomyces coelicolor/genética , Transativadores/genéticaRESUMO
A total of 197 isolates of Staphylococcus from small wild animals (insectivores and rodents) were identified by partial sequencing of the rpoB and dnaJ genes. Among the identified isolates the predominant species was S. succinus (28%), followed by S. xylosus (20.8%) and S. stepanovicii (18.3%). The other 14 Staphylococcus species were occasionally isolated. PCR-RFLP of the rpoB gene digested by Hpy8I was a fast and simple method to distinguish the two subspecies of S. succinus. More than 90% of the 55 S. succinus strains isolated belonged to S. succinus subsp. casei and only 9% to S. succinus subsp. succinus. Moreover, the present study describes the first ever isolation of S. fleurettii from healthy animals.