RESUMO
Spinach is an in vitro-selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.
Assuntos
Quadruplex G , RNA/química , Sequência de Bases , Compostos de Benzil/química , Compostos de Benzil/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Fluorescência , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde , Ligação de Hidrogênio , Imidazolinas/química , Imidazolinas/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Plantas/química , Spinacia oleracea/genéticaRESUMO
The post-translational modification (PTM) of proteins by O-linked ß-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.
RESUMO
Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disorder characterized by differences in autoantibody profiles, serum cytokines, and clinical manifestations. We have previously conducted a case-case genome-wide association study (GWAS) of SLE patients to detect associations with autoantibody profile and serum interferon alpha (IFN-α). In this study, we used public gene expression data sets to rationally select additional single nucleotide polymorphisms (SNPs) for validation. The top 200 GWAS SNPs were searched in a database which compares genome-wide expression data to genome-wide SNP genotype data in HapMap cell lines. SNPs were chosen for validation if they were associated with differential expression of 15 or more genes at a significance of P < 9 × 10(-5). This resulted in 11 SNPs which were genotyped in 453 SLE patients and 418 matched controls. Three SNPs were associated with SLE-associated autoantibodies, and one of these SNPs was also associated with serum IFN-α (P < 4.5 × 10(-3) for all). One additional SNP was associated exclusively with serum IFN-α. Case-control analysis was insensitive to these molecular subphenotype associations. This study illustrates the use of gene expression data to rationally select candidate loci in autoimmune disease, and the utility of stratification by molecular phenotypes in the discovery of additional genetic associations in SLE.
Assuntos
Autoanticorpos/genética , Perfilação da Expressão Gênica , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Polimorfismo de Nucleotídeo Único , Autoanticorpos/imunologia , Linhagem Celular , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Interferon-alfa/sangue , FenótipoRESUMO
Structured RNA elements within the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome hijack host cell machinery for translation initiation through a cap-independent mechanism. Here, using a phage display selection, we obtained two antibody fragments (Fabs), HCV2 and HCV3, against HCV IRES that bind the RNA with dissociation constants of 32 ± 7 nM and 37 ± 8 nM respectively, specifically recognizing the so-called junction IIIabc (JIIIabc). We used these Fabs as crystallization chaperones and determined the high-resolution crystal structures of JIIIabc-HCV2 and -HCV3 complexes at 1.81 Å and 2.75 Å resolution respectively, revealing an antiparallel four-way junction with the IIIa and IIIc subdomains brought together through tertiary interactions. The RNA conformation observed in the structures supports the structural model for this region derived from cryo-EM data for the HCV IRES-40S ribosome complex, suggesting that the tertiary fold of the RNA preorganizes the domain for interactions with the 40S ribosome. Strikingly, both Fabs and the ribosomal protein eS27 not only interact with a common subset of nucleotides within the JIIIabc but also use physiochemically similar sets of protein residues to do so, suggesting that the RNA surface is well-suited for interactions with proteins, perhaps analogous to the "hot spot" concept elaborated for protein-protein interactions. Using a rabbit reticulocyte lysate-based translation assay with a bicistronic reporter construct, we further demonstrated that Fabs HCV2 and HCV3 specifically inhibit the HCV IRES-directed translation, implicating disruption of the JIIIabc-ribosome interaction as a potential therapeutic strategy against HCV.
Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Fragmentos de Imunoglobulinas/química , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Viral/química , Animais , Sequência de Bases , Humanos , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Coelhos , Reticulócitos/metabolismo , Proteínas Ribossômicas/metabolismo , Relação Estrutura-AtividadeRESUMO
Picornaviral IRES elements are essential for initiating the cap-independent viral translation. However, three-dimensional structures of these elements remain elusive. Here, we report a 2.84-Å resolution crystal structure of hepatitis A virus IRES domain V (dV) in complex with a synthetic antibody fragment-a crystallization chaperone. The RNA adopts a three-way junction structure, topologically organized by an adenine-rich stem-loop motif. Despite no obvious sequence homology, the dV architecture shows a striking similarity to a circularly permuted form of encephalomyocarditis virus J-K domain, suggesting a conserved strategy for organizing the domain architecture. Recurrence of the motif led us to use homology modeling tools to compute a 3-dimensional structure of the corresponding domain of foot-and-mouth disease virus, revealing an analogous domain organizing motif. The topological conservation observed among these IRESs and other viral domains implicates a structured three-way junction as an architectural scaffold to pre-organize helical domains for recruiting the translation initiation machinery.
Assuntos
Sequência Conservada , Sítios Internos de Entrada Ribossomal/fisiologia , Motivos de Nucleotídeos/fisiologia , Picornaviridae/fisiologia , RNA Viral/química , RNA Viral/fisiologia , Sequência de Bases , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/metabolismo , Vírus da Hepatite A , Sítios Internos de Entrada Ribossomal/imunologia , Chaperonas Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Viral/metabolismo , Ribossomos/química , Ribossomos/metabolismoRESUMO
BACKGROUND: In systemic lupus erythematosus (SLE), antibodies directed at RNA-binding proteins (anti-RBP) are associated with high serum type I interferon (IFN), which plays an important role in SLE pathogenesis. African-Americans (AA) are more likely to develop SLE, and SLE is also more severe in this population. We hypothesized that peripheral blood gene expression patterns would differ between AA and European-American (EA) SLE patients, and between those with anti-RBP antibodies and those who lack these antibodies. METHODS: Whole blood RNA from 33 female SLE patients and 16 matched female controls from AA and EA ancestral backgrounds was analyzed on Affymetrix Gene 1.0 ST gene expression arrays. Ingenuity Pathway Analysis was used to compare the top differentially expressed canonical pathways amongst the sample groups. An independent cohort of 116 SLE patients was used to replicate findings using quantitative real-time PCR (qPCR). RESULTS: Both AA and EA patients with positive anti-RBP antibodies showed over-expression of similar IFN-related canonical pathways, such as IFN Signaling (P = 1.3 × 10(-7) and 6.3 × 10(-11) in AA vs. EA respectively), Antigen Presenting Pathway (P = 1.8 × 10(-5) and 2.5 × 10(-6)), and a number of pattern recognition receptor pathways. In anti-RBP negative (RBP-) patients, EA subjects demonstrated similar IFN-related pathway activation, whereas no IFN-related pathways were detected in RBP-AA patients. qPCR validation confirmed similar results. CONCLUSION: Our data show that IFN-induced gene expression is completely dependent on the presence of autoantibodies in AA SLE patients but not in EA patients. This molecular heterogeneity suggests differences in IFN-pathway activation between ancestral backgrounds in SLE. This heterogeneity may be clinically important, as therapeutics targeting this pathway are being developed.
RESUMO
All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.
Assuntos
Anticorpos Catalíticos/química , RNA Polimerases Dirigidas por DNA/química , Fragmentos Fab das Imunoglobulinas/química , RNA Catalítico/química , Ribonucleotídeos/química , Catálise , Domínio Catalítico , Conformação de Ácido Nucleico , Biblioteca de Peptídeos , Polinucleotídeo Ligases/química , Proteínas Recombinantes/química , Ribonucleoproteínas/químicaRESUMO
RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab-ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs' high molecular weight, large surface area and phasing power.
Assuntos
Cristalização/métodos , Fragmentos Fab das Imunoglobulinas/química , RNA Catalítico/química , Sequência de Bases , Sítios de Ligação , Ligases/química , Ligases/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biblioteca de Peptídeos , RNA Catalítico/imunologia , RNA não Traduzido/químicaRESUMO
Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.
Assuntos
RNA Catalítico/química , Pareamento de Bases , Sequência de Bases , Catálise , Domínio Catalítico , Cristalização , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Magnésio/química , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Polinucleotídeo Ligases/química , Polinucleotídeo Ligases/metabolismo , RNA Catalítico/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismoRESUMO
Olefin complexes (silox)(3)M(ole) (silox = (t)Bu(3)SiO; M = Nb (1-ole), Ta (2-ole); ole = C(2)H(4), C(2)H(3)Me, C(2)H(3)Et, C(2)H(3)C(6)H(4)-p-X (X = OMe, H, CF(3)), C(2)H(3)(t)Bu, (c)C(5)H(8), (c)C(6)H(10), (c)C(7)H(10) (norbornene)) rearrange to alkylidene isomers (silox)(3)M(alk) (M = Nb (1=alk), Ta (2=alk); alk = CHMe, CHEt, CH(n)Pr, CHCH(2)C(6)H(4)-p-X (X = OMe, H, CF(3) (Ta only)), CHCH(2)(t)Bu, (c)C(5)H(8), (c)C(6)H(10), (c)C(7)H(10) (norbornylidene)). Kinetics and labeling experiments suggest that the rearrangement proceeds via a delta-abstraction on a silox CH bond by the beta-olefin carbon to give (silox)(2)RM(kappa(2)-O,C-OSi(t)Bu(2)CMe(2)CH(2)) (M = Nb (4-R), Ta (6-R); R = Me, Et, (n)Pr, (n)Bu, CH(2)CH(2)C(6)H(4)-p-X (X = OMe, H, CF(3) (Ta only)), CH(2)CH(2)(t)Bu, (c)C(5)H(9), (c)C(6)H(11), (c)C(7)H(11) (norbornyl)). A subsequent alpha-abstraction by the cylometalated "arm" of the intermediate on an alpha-CH bond of R generates the alkylidene 1=alk or 2=alk. Equilibrations of 1-ole with ole' to give 1-ole' and ole, and relevant calculations on 1-ole and 2-ole, permit interpretation of all relative ground and transition state energies for the complexes of either metal.