RESUMEN
Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.
Asunto(s)
Exorribonucleasas/metabolismo , Luteoviridae/genética , Mutación , Conformación de Ácido Nucleico , Estabilidad del ARN , ARN Viral/química , ARN Viral/genética , Regiones no Traducidas 3' , Secuencia de Bases , Cristalización , Genoma Viral , Homología de SecuenciaRESUMEN
RNA nanotechnology seeks to create nanoscale machines by repurposing natural RNA modules. The field is slowed by the current need for human intuition during three-dimensional structural design. Here, we demonstrate that three distinct problems in RNA nanotechnology can be reduced to a pathfinding problem and automatically solved through an algorithm called RNAMake. First, RNAMake discovers highly stable single-chain solutions to the classic problem of aligning a tetraloop and its sequence-distal receptor, with experimental validation from chemical mapping, gel electrophoresis, solution X-ray scattering and crystallography with 2.55 Å resolution. Second, RNAMake automatically generates structured tethers that integrate 16S and 23S ribosomal RNAs into single-chain ribosomal RNAs that remain uncleaved by ribonucleases and assemble onto messenger RNA. Third, RNAMake enables the automated stabilization of small-molecule binding RNAs, with designed tertiary contacts that improve the binding affinity of the ATP aptamer and improve the fluorescence and stability of the Spinach RNA in cell extracts and in living Escherichia coli cells.
Asunto(s)
ARN/química , Cristalografía por Rayos X , Escherichia coli/química , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN de Planta/química , ARN Ribosómico 16S/química , ARN Ribosómico 23S/química , Spinacia oleracea/químicaRESUMEN
Structured RNA elements, programmed RNA conformational changes, and interactions between different RNA domains underlie many modes of regulating gene expression, mandating studies to understand the foundational principles that govern these phenomena. Exploring the structured 3' untranslated region (UTR) of a viral RNA, we discovered that different contexts of the 3'-UTR confer different abilities to enhance translation of an associated open reading frame. In one context, ribosome-induced conformational changes in a 'sensor' RNA domain affect a separate RNA 'functional' domain, altering translation efficiency. The structure of the entire 3'-UTR reveals that structurally distinct domains use a spine of continuously stacked bases and a strut-like linker to create a conduit for communication within the higher-order architecture. Thus, this 3'-UTR RNA illustrates how RNA can use programmed conformational changes to sense the translation status of an upstream open reading frame, then create a tuned functional response by communicating that information to other RNA elements.
Asunto(s)
Regiones no Traducidas 3'/genética , ARN Ribosómico/genética , Regiones no Traducidas 5'/genética , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Viral/genéticaRESUMEN
Folded RNA elements that block processive 5' â 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
Asunto(s)
Exorribonucleasas/metabolismo , Flavivirus/genética , Interacciones Huésped-Patógeno/genética , Pliegue del ARN/genética , ARN Viral/genética , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Conformación de Ácido Nucleico , Estabilidad del ARN/genéticaRESUMEN
The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts to understand the molecular processes of infection. Related flaviviruses produce noncoding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in fetal mice. These viruses make sfRNAs by co-opting a cellular exonuclease via structured RNAs called xrRNAs. We found that ZIKV-infected monkey and human epithelial cells, mouse neurons, and mosquito cells produce sfRNAs. The RNA structure that is responsible for ZIKV sfRNA production forms a complex fold that is likely found in many pathogenic flaviviruses. Mutations that disrupt the structure affect exonuclease resistance in vitro and sfRNA formation during infection. The complete ZIKV xrRNA structure clarifies the mechanism of exonuclease resistance and identifies features that may modulate function in diverse flaviviruses.
Asunto(s)
Exorribonucleasas/química , ARN no Traducido/química , ARN Viral/química , Infección por el Virus Zika/virología , Virus Zika/metabolismo , Animales , Chlorocebus aethiops , Culicidae/virología , Células Epiteliales/virología , Exorribonucleasas/genética , Humanos , Ratones , Mutación , Neuronas/virología , Conformación de Ácido Nucleico , ARN no Traducido/genética , ARN Viral/genética , Células Vero , Virus Zika/genéticaRESUMEN
The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.
Asunto(s)
Bacterias/genética , Eucariontes/genética , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/genética , ARN/química , ARN/genética , Ribosomas/metabolismo , Secuencia de Bases , Secuencia Conservada/genética , Cristalografía por Rayos X , Dicistroviridae/genética , Modelos Moleculares , Iniciación de la Cadena Peptídica Traduccional/genética , ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Ribosomas/químicaRESUMEN
RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles, and this can be conferred by a complex three-dimensional structure. Such multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. To address this gap it is useful to study examples from single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3' end of the turnip yellow mosaic virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic (g)RNA, but also interacts with other structures in the 3' untranslated region of the gRNA, contains the promoter for negative-strand synthesis, and influences several infection-critical processes. TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is 'two faced': one face closely mimics tRNA and drives aminoacylation, the other face diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes.
Asunto(s)
Imitación Molecular , Conformación de Ácido Nucleico , ARN de Transferencia/química , ARN Viral/química , ARN Viral/metabolismo , Tymovirus/genética , Regiones no Traducidas 3' , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Secuencia de Bases , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue del ARN , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Viral/genética , Ribosomas/química , Ribosomas/metabolismo , ARN Pequeño no TraducidoRESUMEN
Flaviviruses are emerging human pathogens and worldwide health threats. During infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) are produced by resisting degradation by the 5'â3' host cell exonuclease Xrn1 through an unknown RNA structure-based mechanism. Here, we present the crystal structure of a complete Xrn1-resistant flaviviral RNA, which contains interwoven pseudoknots within a compact structure that depends on highly conserved nucleotides. The RNA's three-dimensional topology creates a ringlike conformation, with the 5' end of the resistant structure passing through the ring from one side of the fold to the other. Disruption of this structure prevents formation of sfRNA during flaviviral infection. Thus, sfRNA formation results from an RNA fold that interacts directly with Xrn1, presenting the enzyme with a structure that confounds its helicase activity.
Asunto(s)
Virus de la Encefalitis del Valle Murray/genética , Conformación de Ácido Nucleico , ARN Viral/química , Emparejamiento Base , Secuencia de Bases , Cristalografía por Rayos X , Virus de la Encefalitis del Valle Murray/patogenicidad , Exorribonucleasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA ⢠mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.
Asunto(s)
ARN Viral/metabolismo , Ribosomas/metabolismo , Secuencia de Bases , Cristalización , Modelos Moleculares , Conformación de Ácido Nucleico , Filogenia , ARN Viral/químicaRESUMEN
Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.
Asunto(s)
Aniones/metabolismo , ARN/química , ARN/metabolismo , Aminoácidos/metabolismo , Aniones/química , Sitios de Unión , Cationes/química , Cationes/metabolismo , Cristalografía , Modelos Moleculares , Conformación de Ácido Nucleico , Proteínas/química , Proteínas/metabolismo , Ácido Selénico , Compuestos de Selenio/química , Compuestos de Selenio/metabolismo , Sulfatos/química , Sulfatos/metabolismo , Difracción de Rayos XRESUMEN
Internal ribosome entry site (IRES) RNAs initiate protein synthesis in eukaryotic cells by a noncanonical cap-independent mechanism. IRESes are critical for many pathogenic viruses, but efforts to understand their function are complicated by the diversity of IRES sequences as well as by limited high-resolution structural information. The intergenic region (IGR) IRESes of the Dicistroviridae viruses are powerful model systems to begin to understand IRES function. Here we present the crystal structure of a Dicistroviridae IGR IRES domain that interacts with the ribosome's decoding groove. We find that this RNA domain precisely mimics the transfer RNA anticodon-messenger RNA codon interaction, and its modeled orientation on the ribosome helps explain translocation without peptide bond formation. When combined with a previous structure, this work completes the first high-resolution description of an IRES RNA and provides insight into how RNAs can manipulate complex biological machines.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/química , ARN de Transferencia/química , Ribosomas/fisiología , Anticodón/genética , Sitios de Unión , Hepacivirus/genética , Modelos Genéticos , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Ribosomas/genéticaRESUMEN
BACKGROUND: Poland syndrome encompasses a constellation of congenital chest wall, breast, and upper-extremity deformities, which present a significant reconstructive challenge for the plastic surgeon. The broad range of chest wall and breast anomalies has led to an equally broad variety of surgical solutions. Often, however, initial attempts at surgical correction fall short because of failure to identify the best reconstructive option for an individual's particular deformity. METHODS: In a retrospective series of 29 patients, we report our institution's experience with reconstructing breast and chest contour deformities associated with Poland syndrome. We also present a review of the literature. RESULTS: The breast and chest wall deformities associated with Poland syndrome can be effectively treated in an algorithmic, individualized fashion. CONCLUSIONS: We propose an algorithmic approach to the patient with a Poland syndrome chest wall and breast deformity.
Asunto(s)
Mama/cirugía , Mamoplastia/métodos , Procedimientos de Cirugía Plástica , Síndrome de Poland/cirugía , Prótesis e Implantes , Traumatismos de los Tejidos Blandos/cirugía , Cirugía Plástica/métodos , Adolescente , Adulto , Implantación de Mama/métodos , Femenino , Humanos , Masculino , Estudios Retrospectivos , Pared Torácica/patologíaRESUMEN
Internal ribosome entry sites (IRESs) substitute RNA sequences for some or all of the canonical translation initiation protein factors. Therefore, an important component of understanding IRES function is a description of the three-dimensional structure of the IRES RNA underlying this mechanism. This includes determining the degree to which the RNA folds, the global RNA architecture, and higher resolution information when warranted. Knowledge of the RNA structural features guides ongoing mechanistic and functional studies. In this chapter, we present a roadmap to structurally characterize a folded RNA, beginning from initial studies to define the overall architecture and leading to high-resolution structural studies. The experimental strategy presented here is not unique to IRES RNAs but is adaptable to virtually any RNA of interest, although characterization of RNA-protein interactions requires additional methods. Because IRES RNAs have a specific function, we present specific ways in which the data are interpreted to gain insight into that function. We provide protocols for key experiments that are particularly useful for studying IRES RNA structure and that provide a framework onto which additional approaches are integrated. The protocols we present are solution hydroxyl radical probing, RNase T1 probing, native gel electrophoresis, sedimentation velocity analytical ultracentrifugation, and strategies to engineer RNA for crystallization and to obtain initial crystals.
Asunto(s)
Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , ARN/química , ARN/metabolismo , Ribosomas/metabolismo , Secuencia de Bases , Bioensayo/métodos , Radical Hidroxilo/química , Sondas Moleculares/química , Datos de Secuencia Molecular , Mutación , ARN/genética , Ribosomas/químicaRESUMEN
Internal ribosome entry site (IRES) RNAs are necessary for successful infection of many pathogenic viruses, but the details of the RNA structure-based mechanism used to bind and manipulate the ribosome remain poorly understood. The IRES RNAs from the Dicistroviridae intergenic region (IGR) are an excellent model system to understand the fundamental tenets of IRES function, requiring no protein factors to manipulate the ribosome and initiate translation. Here, we explore the architecture of four members of the IGR IRESes, representative of the two divergent classes of these IRES RNAs. Using biochemical and structural probing methods, we show that despite sequence variability they contain a common three-dimensional fold. The three-dimensional architecture of the ribosome binding domain from these IRESes is organized around a core helical scaffold, around which the rest of the RNA molecule folds. However, subtle variation in the folds of these IRESes and the presence of an additional secondary structure element suggest differences in the details of their manipulation of the large ribosomal subunit. Overall, the results demonstrate how a conserved three-dimensional RNA fold governs ribosome binding and manipulation.
Asunto(s)
Modelos Moleculares , Conformación de Ácido Nucleico , Virus ARN/genética , ARN Viral/química , Ribosomas/genética , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Canonical cap-dependent translation initiation requires a large number of protein factors that act in a stepwise assembly process. In contrast, internal ribosomal entry sites (IRESs) are cis-acting RNAs that in some cases completely supplant these factors by recruiting and activating the ribosome using a single structured RNA. Here we present the crystal structures of the ribosome-binding domain from a Dicistroviridae intergenic region IRES at 3.1 angstrom resolution, providing a view of the prefolded architecture of an all-RNA translation initiation apparatus. Docking of the structure into cryo-electron microscopy reconstructions of an IRES-ribosome complex suggests a model for ribosome manipulation by a dynamic IRES RNA.
Asunto(s)
Biosíntesis de Proteínas , Virus ARN/genética , ARN Viral/química , Secuencias Reguladoras de Ácido Ribonucleico , Ribosomas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , ARN Viral/genética , ARN Viral/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico/genéticaRESUMEN
Basosquamous carcinoma of the skin is a relatively rare cutaneous neoplasm that has been shown to have significant metastatic potential. Histopathologists debate whether these lesions arise de novo or differentiate from pre-existing basal cell carcinomas. We present a case in which a longstanding lesion initially diagnosed as basal cell carcinoma was later found to have basosquamous histology and regional metastases. Review of the literature reveals a metastatic rate greater than that of basal cell and squamous cell carcinoma, and identifies several important characteristics that impact prognosis after surgical resection. For physicians treating carcinomas of the skin, it is important to understand the natural history and proper treatment of this aggressive neoplasm.
Asunto(s)
Carcinoma Basoescamoso/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Enfermedades del Pie/diagnóstico , Neoplasias Cutáneas/diagnóstico , Diagnóstico Diferencial , Humanos , Metástasis Linfática/diagnóstico , Masculino , Persona de Mediana EdadRESUMEN
Proopiomelanocortin (POMC) cDNAs were cloned and sequenced from brain extracts of two species of urodele amphibians: Amphiuma means and Necturus maculosus. Although the two species of urodele amphibians belong to separate families, and do not share a direct common ancestor, the level of primary sequence identity for the open reading of the POMC cDNAs was 90% at the amino acid level and 79% at the nucleotide level. It appears that the POMC gene in these urodele amphibians has been accumulating mutations at the amino acid level at a slower rate than the POMC gene in other sarcopterygian orders.
Asunto(s)
Evolución Molecular , Necturus maculosus/genética , Proopiomelanocortina/genética , Proopiomelanocortina/efectos de la radiación , Urodelos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Hipófisis/metabolismo , Hipófisis/efectos de la radiación , Proopiomelanocortina/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , gamma-MSH/genética , gamma-MSH/efectos de la radiaciónRESUMEN
The internal ribosome site RNA of the cricket paralysis-like viruses (CrPV-like) binds directly to the ribosome, assembling the translation machinery without initiation factors. This mechanism does not require initiator tRNA, and translation starts from a non-AUG codon. A wealth of biochemical data has yielded a working model for this process, but the three-dimensional structure and biophysical characteristics of the unbound CrPV-like IRES RNAs are largely unexplored. Here, we demonstrate that the CrPV-like IRESes prefold into a two-part structure in the presence of magnesium ions. The largest part is a prefolded compact RNA domain that shares folding and structural characteristics with other compactly folded RNAs such as group I intron RNAs and RNase P RNA. Chemical probing reveals that the CrPV-like IRES' compact domain contains RNA helices that are packed tightly enough to exclude solvent, and analytical ultracentrifugation indicates a large change in the shape of the IRES upon folding. Formation of this compact domain is necessary for binding of the 40S subunit, and the structural organization of the unbound IRES RNA is consistent with the hypothesis that the IRES is functionally and structurally preorganized before ribosome binding.