RESUMEN
Recent ribosome profiling and proteomic studies have revealed the presence of thousands of novel coding sequences, referred to as small open reading frames (sORFs), in prokaryotic and eukaryotic genomes. These genes have defied discovery via traditional genomic tools not only because they tend to be shorter than standard gene annotation length cutoffs, but also because they are, as a class, enriched in sequence properties previously assumed to be unusual, including non-AUG start codons. In this review, we summarize what is currently known about the incidence, efficiency, and mechanism of non-AUG start codon usage in prokaryotes and eukaryotes, and provide examples of regulatory and functional sORFs that initiate at non-AUG codons. While only a handful of non-AUG-initiated novel genes have been characterized in detail to date, their participation in important biological processes suggests that an improved understanding of this class of genes is needed.
Asunto(s)
Codón Iniciador/química , Genoma , Sistemas de Lectura Abierta , Iniciación de la Cadena Peptídica Traduccional , Proteoma/genética , Ribosomas/genética , Codón Iniciador/metabolismo , Biología Computacional/métodos , Eucariontes/genética , Eucariontes/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular/métodos , Células Procariotas/metabolismo , Señales de Clasificación de Proteína/genética , Proteoma/clasificación , Proteoma/metabolismo , Ribosomas/clasificación , Ribosomas/metabolismoRESUMEN
Growing evidence illustrates the shortcomings on the current understanding of the full complexity of the proteome. Previously overlooked small open reading frames (sORFs) and their encoded microproteins have filled important gaps, exerting their function as biologically relevant regulators. The characterization of the full small proteome has potential applications in many fields. Continuous development of techniques and tools led to an improved sORF discovery, where these can originate from bioinformatics analyses, from sequencing routines or proteomics approaches. In this mini review, we discuss the ongoing trends in the three fields and suggest some strategies for further characterization of high potential candidates.
Asunto(s)
Biología Computacional/estadística & datos numéricos , Redes Neurales de la Computación , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Proteoma/genética , Ribosomas/genética , Animales , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Plantas/genética , Señales de Clasificación de Proteína/genética , Proteoma/clasificación , Proteoma/metabolismo , Ribosomas/clasificación , Ribosomas/metabolismo , Programas InformáticosRESUMEN
High throughput RNA sequencing techniques have revealed that a large fraction of the genome is transcribed into long non-coding RNAs (lncRNAs). Unlike canonical protein-coding genes, lncRNAs do not contain long open reading frames (ORFs) and tend to be poorly conserved across species. However, many of them contain small ORFs (sORFs) that exhibit translation signatures according to ribosome profiling or proteomics data. These sORFs are a source of putative novel proteins; some of them may confer a selective advantage and be maintained over time, a process known as de novo gene birth. Here we review the mechanisms by which randomly occurring sORFs in lncRNAs can become new functional proteins.
Asunto(s)
Evolución Molecular , Genoma , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Largo no Codificante/genética , Ribosomas/genética , Animales , Encéfalo/metabolismo , Humanos , Hígado/metabolismo , Masculino , Anotación de Secuencia Molecular , Miocardio/metabolismo , Especificidad de Órganos , ARN Largo no Codificante/clasificación , ARN Largo no Codificante/metabolismo , Ribosomas/clasificación , Ribosomas/metabolismo , Testículo/metabolismo , Transcripción GenéticaRESUMEN
It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gain-of-function assays and the demonstration of inducibility under physiological conditions.
Asunto(s)
Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN de Transferencia/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Animales , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Secuencia de Bases , Heterogeneidad Genética , Mamíferos/genética , Mamíferos/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/clasificación , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The ribosome exit tunnel is an important structure involved in the regulation of translation and other essential functions such as protein folding. By comparing 20 recently obtained cryo-EM and X-ray crystallography structures of the ribosome from all three domains of life, we here characterize the key similarities and differences of the tunnel across species. We first show that a hierarchical clustering of tunnel shapes closely reflects the species phylogeny. Then, by analyzing the ribosomal RNAs and proteins, we explain the observed geometric variations and show direct association between the conservations of the geometry, structure and sequence. We find that the tunnel is more conserved in the upper part close to the polypeptide transferase center, while in the lower part, it is substantially narrower in eukaryotes than in bacteria. Furthermore, we provide evidence for the existence of a second constriction site in eukaryotic exit tunnels. Overall, these results have several evolutionary and functional implications, which explain certain differences between eukaryotes and prokaryotes in their translation mechanisms. In particular, they suggest that major co-translational functions of bacterial tunnels were externalized in eukaryotes, while reducing the tunnel size provided some other advantages, such as facilitating the nascent chain elongation and enabling antibiotic resistance.
Asunto(s)
Archaea/genética , Bacterias/genética , Eucariontes/genética , Biosíntesis de Proteínas , ARN Ribosómico/química , Proteínas Ribosómicas/química , Ribosomas/ultraestructura , Secuencia de Aminoácidos , Archaea/clasificación , Archaea/metabolismo , Bacterias/clasificación , Bacterias/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Eucariontes/clasificación , Eucariontes/metabolismo , Conformación de Ácido Nucleico , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/clasificación , Ribosomas/genética , Ribosomas/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Ribosomes associated with nonsense-mediated decay factors Upf1, Upf2, or Upf3 were purified by immunoprecipitation, and enrichment and stoichiometry of Upf factors and ribosomal proteins were analyzed by western blot and mass spectrometry. Using a small RNA library preparation protocol that eliminates in-gel RNA and cDNA size selection and incorporates four random nucleotides on each side of the ribosome-protected RNA fragment allowed recovery, detection, and analysis of all size classes of protected fragments from a sample simultaneously.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN Helicasas/genética , ARN de Hongos/genética , ARN Mensajero/genética , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Codón sin Sentido , Inmunoprecipitación/métodos , Degradación de ARNm Mediada por Codón sin Sentido , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/clasificación , Ribosomas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosomal proteins (r-proteins) are increasingly used as an alternative to ribosomal rRNA for prokaryotic systematics. However, their routine use is difficult because r-proteins are often not or wrongly annotated in complete genome sequences, and there is currently no dedicated exhaustive database of r-proteins. RiboDB aims at fulfilling this gap. This weekly updated comprehensive database allows the fast and easy retrieval of r-protein sequences from publicly available complete prokaryotic genome sequences. The current version of RiboDB contains 90 r-proteins from 3,750 prokaryotic complete genomes encompassing 38 phyla/major classes and 1,759 different species. RiboDB is accessible at http://ribodb.univ-lyon1.fr and through ACNUC interfaces.
Asunto(s)
Bases de Datos Factuales , Proteínas Ribosómicas/clasificación , Secuencia de Bases , Bases de Datos de Proteínas , Filogenia , Células Procariotas/clasificación , ARN Ribosómico , Ribosomas/clasificación , Programas InformáticosRESUMEN
The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed the native structure and organization of the cytoplasmic translation machinery. Analysis of a large dynamic structure-the nuclear pore complex-revealed variations detectable at the level of individual complexes. Cryo-ET was used to visualize previously elusive structures, such as nucleosome chains and the filaments of the nuclear lamina, in situ. Elucidation of the lamina structure provides insight into its contribution to metazoan nuclear stiffness.
Asunto(s)
Lámina Nuclear/ultraestructura , Poro Nuclear/ultraestructura , Microscopía por Crioelectrón/métodos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Células HeLa , Humanos , Nucleosomas/ultraestructura , Biosíntesis de Proteínas , Ribosomas/clasificación , Ribosomas/ultraestructuraRESUMEN
Single molecule methods have revealed that heterogeneity is common in biological systems. However, interpretations of the complex signals are challenging. By tracking the fluorescence resonance energy transfer (FRET) signals between the A-site tRNA and L27 protein in single ribosomes, we attempt to develop a qualitative method to subtract the inherent patterns of the heterogeneous single molecule FRET data. Seven ribosome subpopulations are identified using this method and spontaneous exchanges among these subpopulations are observed. All of the pretranslocation subpopulations are competent in real-time translocation, but via distinguished pathways. These observations suggest that the ribosome may function through multiple reaction pathways.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Biosíntesis de Proteínas/fisiología , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/fisiología , Ribosomas/ultraestructura , Transducción de Señal/fisiología , Ribosomas/clasificaciónRESUMEN
In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the 'hibernating ribosome'. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF.
Asunto(s)
Bacterias/química , Evolución Molecular , Ribosomas/química , Ribosomas/clasificación , Bacterias/metabolismo , Proteínas Ribosómicas/análisis , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.
Asunto(s)
Codón de Terminación/fisiología , Biosíntesis de Proteínas/fisiología , Ribosomas/fisiología , Ribosomas/clasificación , Factores de TiempoRESUMEN
TA cloning methods are widely used in analyses of environmental microbial diversity, yet the potential of TA methods to yield phylogenetically biased results has received little attention. To test for a TA bias, we constructed clone libraries of fungal amplicons spanning the ribosomal internally transcribed spacer (ITS) and partial large subunit (LSU) from 92 boreal forest soil DNA extracts using two contrasting methods: the Invitrogen TOPO-TA system and the Lucigen PCR-SMART system. The Lucigen system utilizes blunt-ended rather than TA cloning and transcription terminators to reduce biases due to toxicity of expressed inserts. We analysed 588 clone sequences from the two libraries. Species diversity estimators applied to operational taxonomical units (OTUs) were slightly higher for Invitrogen than Lucigen, but confidence intervals for accumulation curves overlapped. Abundances of OTUs were correlated between the libraries (r(2) = 0.5, P < 0.0001), but certain OTUs had contrasting abundances in the two libraries and a likelihood ratio test rejected homogeneity of the OTU counts. We constructed parsimony and Bayesian trees from aligned LSU regions, and the 'phylogenetic test' revealed that lineage representation was not significantly different between the two libraries. We conclude that characterization of this fungal community was fairly robust to cloning method and no biases due to TA cloning were found.
Asunto(s)
Clonación Molecular/métodos , Hongos/genética , Biblioteca de Genes , Ribosomas/clasificación , Análisis de Secuencia de ADN/instrumentación , Microbiología del Suelo , FilogeniaRESUMEN
A method of computing correlation coefficients for object detection that takes advantage of using azimuthally averaged reference projections is described and compared with two alternative methods-computing a cross-correlation function or a local correlation coefficient versus the azimuthally averaged reference projections. Two examples of an application from structural biology involving the detection of projection views of biological macromolecules in electron micrographs are discussed. It is found that a novel approach to computing a local correlation coefficient versus azimuthally averaged reference projections, using a rotational correlation coefficient, outperforms using a cross-correlation function and a local correlation coefficient in object detection from simulated images with a range of levels of simulated additive noise. The three approaches perform similarly in detecting macromolecular views in electron microscope images of a globular macrolecular complex (the ribosome). The rotational correlation coefficient outperforms the other methods in detection of keyhole limpet hemocyanin macromolecular views in electron micrographs.
Asunto(s)
Algoritmos , Inteligencia Artificial , Microscopía por Crioelectrón/métodos , Interpretación de Imagen Asistida por Computador/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Ribosomas/ultraestructura , Técnica de Sustracción , Gráficos por Computador , Aumento de la Imagen/métodos , Almacenamiento y Recuperación de la Información/métodos , Análisis Numérico Asistido por Computador , Reproducibilidad de los Resultados , Ribosomas/clasificación , Sensibilidad y Especificidad , Estadística como AsuntoRESUMEN
Based on phylogenetic analysis of rDNA internal transcribed spacer (ITS) sequences, a pair of specific primers were designed for differentiating the Chinese traditional medicine Hericium species from other mushrooms by PCR. PCR was performed, with total DNAs as a template at an annealing temperature of 52-57 degrees C. Positive amplification was obtained from H. erinaceus with all DNA templates from different resources, but not from other related species. The result indicated that H. erinaceus could be clearly distinguished from other fungi by detection PCR, and no incorrect discrimination was found under the same reaction conditions. The primers were also successfully employed to identify H. erinaceus with different tissue types.
Asunto(s)
Agaricales/genética , ADN Espaciador Ribosómico/genética , Hongos/genética , Reacción en Cadena de la Polimerasa/métodos , Ribosomas/genética , Agaricales/clasificación , Secuencia de Aminoácidos/genética , Hongos/clasificación , Datos de Secuencia Molecular , Filogenia , Plantas Medicinales/clasificación , Plantas Medicinales/genética , Ribosomas/clasificaciónRESUMEN
The human malaria parasite Plasmodium vivax has been shown to regulate the transcription of two distinct 18 RNAs during development. Here we show a third and distinctive type of ribosome that is present shortly after zygote formation, a transcriptional pattern of ribosome types that relates closely to the developmental state of the parasite and a phenomenon that separates ribosomal types at a critical phase of maturation. The A-type ribosome is predominantly found in infected erythrocytes of the vertebrate and the mosquito blood meal. Transcripts from the A gene are replaced by transcripts from another locus, the O gene, shortly after fertilization and increase in number as the parasite develops on the mosquito midgut. Transcripts from another locus, the S gene, begins as the oocyst form of the parasite matures. RNA transcripts from the S gene are preferentially included in sporozoites that bud off from the oocyst and migrate to the salivary gland while the O gene transcripts are left within the oocyst. Although all three genes are typically eukaryotic in structure, the O gene transcript, described here, varies from the other two in core regions of the rRNA that are involved in mRNA decoding and translational termination. We now can correlate developmental progression of the parasite with changes in regions of rRNA sequence that are broadly conserved, where sequence alterations have been related to function in other systems and whose effects can be studied outside of Plasmodium. This should allow assessment of the role of translational control in parasite development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios , Plasmodium vivax/crecimiento & desarrollo , ARN Ribosómico 18S/genética , Ribosomas/genética , Animales , Anopheles/parasitología , Secuencia de Bases , Eritrocitos/parasitología , Humanos , Malaria Vivax/parasitología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Plasmodium vivax/clasificación , Plasmodium vivax/genética , Biosíntesis de Proteínas , ARN Protozoario/biosíntesis , ARN Ribosómico 18S/biosíntesis , ARN Ribosómico 18S/clasificación , Ribosomas/clasificación , Homología de Secuencia de Ácido Nucleico , CigotoRESUMEN
The aim of this work was to develop and evaluate a molecular typing strategy for Salmonella based on hybridization of chromosomal DNA with two different probes derived from insertion sequence IS200. Probe IS200-TT was specifically constructed for this study as a trimer of a 112 pb TaqI-TaqI fragment of IS200. Among several restriction enzymes evaluated, two were selected: EcoRI, which cuts the insertion sequence in two pieces, each one complementary to one of the probes used, and PstI, a restriction enzyme with no recognition site into IS200. With several combinations of these restrictions enzymes and probes, 43 Salmonella typhimurium strains were analyzed for copy number and location of IS200, as well as reproducibility and stability of the patterns. IS200 types have been shown to be stable, both in strains isolated from different patients implicated in the same salmonellosis outbreak and in strains isolated from the same patient at different times or from different specimens. The discriminatory power of the method has been 0.91 to 0.94. As a comparison, S. typhimurium strains were also ribotyped. Discriminatory power of the ribotypes oscillated between 0.44 and 0.55, depending on the enzyme used, and achieved a 0.78 value when the information obtained with two restriction enzymes was combined. Moreover, IS200 typing was able to differentiate among a group of S. typhimurium strains which were identical by ribotype and enzymatic electrophoretic mobility. These results enable us to conclude that, for the stability, reproducibility and discriminatory power of the patterns generated, IS200 probes can be a very useful tool in the molecular typing of S. typhimurium.