RESUMEN

Here, we employ coelution experiments and far-western blotting to identify stable interactions between the main components of the B. subtilis degradosome and the small proteins SR1P and SR7P. Our data indicate that B. subtilis has a degradosome comprising at least RNases Y and PnpA, enolase, phosphofructokinase, glycerol-3-phosphate dehydrogenase GapA, and helicase CshA that can be co-purified without cross-linking. All interactions were corroborated by far-western blotting with proteins purified from E. coli. Previously, we discovered that stress-induced SR7P binds enolase to enhance its interaction with and activity of enolase-bound RNase Y (RnY), while SR1P transcribed under gluconeogenic conditions interacts with GapA to stimulate its interaction with and the activity of RnjA (RnjA). We show that SR1P can directly bind RnjA, RnY, and PnpA independently of GapA, whereas SR7P only interacts with enolase. Northern blotting suggests that the degradation of individual RNAs in B. subtilis under gluconeogenic or stress conditions depends on either RnjA or RnY alone or on RnjA-SR1P, RnY-SR1P, or RnY-Eno. In vitro degradation assays with RnY or RnjA substrates corroborate the in vivo role of SR1P. Currently, it is unknown which substrate property is decisive for the utilization of one of the complexes.

Asunto(s)

Bacillus subtilis , Escherichia coli , Complejos Multienzimáticos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Endorribonucleasas/metabolismo , ARN Helicasas/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Fosfopiruvato Hidratasa/genética , Fosfopiruvato Hidratasa/metabolismo

RESUMEN

It is widely accepted that the earliest RNA molecules were folded into hairpins or mini-helixes. Herein, we depict the 2D and 3D conformations of those earliest RNA molecules with only RNY triplets, which Eigen proposed as the primeval genetic code. We selected 26 species (13 bacteria and 13 archaea). We found that the free energy of RNY hairpins was consistently lower than that of their corresponding shuffled controls. We found traces of the three ribosomal RNAs (16S, 23S, and 5S), tRNAs, 6S RNA, and the RNA moieties of RNase P and the signal recognition particle. Nevertheless, at this stage of evolution there was no genetic code (as seen in the absence of the peptidyl transferase centre and any vestiges of the anti-Shine-Dalgarno sequence). Interestingly, we detected the anticodons of both glycine (GCC) and threonine (GGU) in the hairpins of proto-tRNA.

Asunto(s)

Evolución Molecular , Código Genético , ARN de Transferencia/genética , ARN/genética , Bacterias/genética

RESUMEN

In this work, we formulate the following question: How the distribution of aminoacyl-tRNA synthetases (aaRSs) went from an ancestral bidirectional gene (mirror symmetry) to the symmetrical distribution of aaRSs in a six-dimensional hypercube of the Standard Genetic Code (SGC)? We assume a primeval RNY code, two Extended Genetic RNA codes type 1 and 2, and the SGC. We outline the types of symmetries of the distribution of aaRSs in each code. The symmetry groups of aaRSs in each code are described, until the symmetries of the SGC display a mirror symmetry. Considering both Extended RNA codes the 20 aaRSs were already present before the Last Universal Ancestor. These findings reveal intricacies in the diversification of aaRSs accompanied by the evolution of the genetic code.

Asunto(s)

Aminoacil-ARNt Sintetasas , Evolución Molecular , Código Genético , Aminoacil-ARNt Sintetasas/genética , ARN de Transferencia/genética , ARN

RESUMEN

Y RNA are a class of small non-coding RNA that are largely conserved. Although their discovery was almost 40 years ago, their function is still under investigation. This is evident in cancer biology, where their role was first studied just a dozen years ago. Since then, only a few contributions were published, mostly scattered across different tumor types and, in some cases, also suffering from methodological limitations. Nonetheless, these sparse data may be used to make some estimations and suggest routes to better understand the role of Y RNA in cancer formation and characterization. Here we summarize the current knowledge about Y RNA in multiple types of cancer, also including a paragraph about tumors that might be included in this list in the future, if more evidence becomes available. The picture arising indicates that Y RNA might be useful in tumor characterization, also relying on non-invasive methods, such as the analysis of the content of extracellular vesicles (EV) that are retrieved from blood plasma and other bodily fluids. Due to the established role of Y RNA in DNA replication, it is possible to hypothesize their therapeutic targeting to inhibit cell proliferation in oncological patients.

RESUMEN

YRNAs are a class of non-coding RNAs that are components of the Ro60 ribonucleoprotein particle and are essential for initiation of DNA replication. Ro60 ribonucleoprotein particle is a target of autoimmune antibodies in patients suffering from systemic lupus erythematosus and Sjögren's syndrome. Deregulation of YRNAs has been confirmed in many cancer types, but not in head and neck squamous cell carcinoma (HNSCC). The main aim of this study was to determine the biological role of YRNAs in HNSCC, the expression of YRNAs, and their usefulness as potential HNSCC biomarkers. Using quantitative reverse transcriptase (qRT)-PCR, the expression of YRNAs was measured in HNSCC cell lines, 20 matched cancer tissues, and 70 FFPETs (Formaline-Fixed Paraffin-Embedded Tissue) from HNSCC patients. Using TCGA (The Cancer Genome Atlas) data, an analysis of the expression levels of selected genes, and clinical-pathological parameters was performed. The expression of low and high YRNA1 expressed groups were analysed using gene set enrichment analysis (GSEA). YRNA1 and YRNA5 are significantly downregulated in HNSCC cell lines. YRNA1 was found to be significantly downregulated in patients' tumour sample. YRNAs were significantly upregulated in T4 stage. YRNA1 showed the highest sensitivity, allowing to distinguish healthy from cancer tissue. An analysis of TCGA data revealed that expression of YRNA1 was significantly altered in the human papilloma virus (HPV) infection status. Patients with medium or high expression of YRNA1 showed better survival outcomes. It was noted that genes correlated with YRNA1 were associated with various processes occurring during cancerogenesis. The GSEA analysis showed high expression enrichment in eight vital processes for cancer development. YRNA1 influence patients' survival and could be used as an HNSCC biomarker. YRNA1 seems to be a good potential biomarker for HNSCC, however, more studies must be performed and these observations should be verified using an in vitro model.

Asunto(s)

Neoplasias de Cabeza y Cuello/genética , ARN no Traducido/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Anciano , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Papillomaviridae/fisiología , ARN no Traducido/genética , Curva ROC , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Resultado del Tratamiento

RESUMEN

The attempt to delineate the essential features that characterized life in its beginnings and the understanding of how those features evolved, represent important scientific challenges. While there have been varied efforts in the elucidation of how the first biomolecules arose from a prebiotic environment, there remains important gaps towards the characterization of the complete repertoire of the Last Universal Common Ancestor (LUCA). We portray a step-wise proteome evolution, by looking at the phenotype encoded by each one of the genetic codes that were appearing along evolution, beginning with the primeval genetic code and then with two intermediate Extended RNA codes, which finally shaped the current Standard Genetic Code (SGC). Notably, all molecules involved in translation, such as ribosomal proteins and all tRNA synthetases, were already present before LUCA. The metabolism belonged to extremophiles as hinted by the presence of reverse gyrase and acetyl coenzyme A synthase. Furthermore, we predict the structure and possible ligands of the proteins retrieved. We have forged a bridge between the hitherto unknown proteome of progenotes and the proteome of LUCA.

Asunto(s)

Evolución Molecular , Código Genético/genética , Proteoma/química , Proteoma/genética , Animales , Humanos , Filogenia , Estructura Secundaria de Proteína

RESUMEN

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.

Asunto(s)

Proteína 4 Similar a ELAV/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Neuronas Motoras/fisiología , ARN Pequeño no Traducido/genética , Regiones no Traducidas 3' , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Línea Celular , Proteína 4 Similar a ELAV/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Neuronas Motoras/metabolismo , Neurogénesis/genética , Polirribosomas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo

RESUMEN

Herein we outline a plausible proteome, encoded by assuming a primeval RNY genetic code. We unveil the primeval phenotype by using only the RNA genotype; it means that we recovered the most ancestral proteome, mostly made of the 8 amino acids encoded by RNY triplets. By looking at those fragments, it is noticeable that they are positioned, not at catalytic sites, but in the cofactor binding sites. It implies that the stabilization of a molecule appeared long before its catalytic activity, and therefore the Ur-proteome comprised a set of proteins modules that corresponded to Cofactor Stabilizing Binding Sites (CSBSs), which we call the primitive bindome. With our method, we reconstructed the structures of the "first protein modules" that Sobolevsky and Trifonov (2006) found by using only RMSD. We also examine the probable cofactors that bound to them. We discuss the notion of CSBSs as the first proteins modules in progenotes in the context of several proposals about the primitive forms of life.

Asunto(s)

Aminoácidos/química , Evolución Molecular , Origen de la Vida , Proteoma/química , ARN/química , Código Genético

RESUMEN

Scaffold proteins are ubiquitous chaperones that bind proteins and facilitate physical interaction of multi-enzyme complexes. Here we used a biochemical approach to dissect the scaffold activity of the flotillin-homolog protein FloA of the multi-drug-resistant human pathogen Staphylococcus aureus. We show that FloA promotes oligomerization of membrane protein complexes, such as the membrane-associated RNase Rny, which forms part of the RNA-degradation machinery called the degradosome. Cells lacking FloA had reduced Rny function and a consequent increase in the targeted sRNA transcripts that negatively regulate S. aureus toxin expression. Small molecules that altered FloA oligomerization also reduced Rny function and decreased the virulence potential of S. aureus in vitro, as well as in vivo, using invertebrate and murine infection models. Our results suggest that flotillin assists in the assembly of protein complexes involved in S. aureus virulence, and could thus be an attractive target for the development of new antimicrobial therapies.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Staphylococcus aureus/patogenicidad , Virulencia , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Femenino , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Multimerización de Proteína/efectos de los fármacos , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Bacteriano/metabolismo , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/mortalidad , Infecciones Estafilocócicas/patología , Staphylococcus aureus/metabolismo , Tasa de Supervivencia , Técnicas del Sistema de Dos Híbridos , Virulencia/efectos de los fármacos , Virulencia/genética

RESUMEN

In this work, we determine the biological and mathematical properties that are sufficient and necessary to uniquely determine both the primeval RNY (purine-any base-pyrimidine) code and the standard genetic code (SGC). These properties are: the evolution of the SGC from the RNY code; the degeneracy of both codes, and the non-degeneracy of the assignments of aminoacyl-tRNA synthetases (aaRSs) to amino acids; the wobbling property; the consideration that glycine was the first amino acid; the topological and symmetrical properties of both codes.

RESUMEN

Most bacterial toxins derived from chromosomally encoded toxin-antitoxin (TA) systems that have been studied to date appear to protect cells from relatively short pulses of stress by triggering a reversible state of growth arrest. In contrast to many bacterial toxins that are produced as defense mechanisms and secreted from their hosts, TA toxins exert their protective effect from within the cell that produces them. TA toxin-mediated growth arrest is most frequently achieved through their ability to selectively cleave RNA species that participate in protein synthesis. Until very recently, it was thought that the primary conduit for toxin-mediated translation inhibition was cleavage of a single class of RNA, mRNA, thus depleting transcripts and precluding production of essential proteins. This minireview focuses on how the development and implementation of a specialized RNA-seq method to study Mycobacterium tuberculosis TA systems enabled the identification of unexpected RNA targets for toxins, i.e. a handful of tRNAs that are cleaved into tRNA halves. Our result brings to light a new perspective on how these toxins may act in this pathogen and uncovers a striking parallel to signature features of the eukaryotic stress response.

Asunto(s)

Antitoxinas/genética , Toxinas Bacterianas/genética , Mycobacterium tuberculosis/genética , ARN Bacteriano/genética , ARN de Transferencia/genética , Tuberculosis/virología , Animales , Antitoxinas/química , Antitoxinas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Colicinas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Secuencias Invertidas Repetidas , Mycobacterium tuberculosis/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismo , Análisis de Secuencia de ARN , Estrés Fisiológico

RESUMEN

Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion.

Asunto(s)

Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , ARN/metabolismo , Comunicación Celular/fisiología , Muerte Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Células K562

RESUMEN

For this report, we analyzed protein secondary structures in relation to the statistics of three nucleotide codon positions. The purpose of this investigation was to find which properties of the ribosome, tRNA or protein level, could explain the purine bias (Rrr) as it is observed in coding DNA. We found that the Rrr pattern is the consequence of a regularity (the codon structure) resulting from physicochemical constraints on proteins and thermodynamic constraints on ribosomal machinery. The physicochemical constraints on proteins mainly come from the hydropathy and molecular weight (MW) of secondary structures as well as the energy cost of amino acid synthesis. These constraints appear through a network of statistical correlations, such as (i) the cost of amino acid synthesis, which is in favor of a higher level of guanine in the first codon position, (ii) the constructive contribution of hydropathy alternation in proteins, (iii) the spatial organization of secondary structure in proteins according to solvent accessibility, (iv) the spatial organization of secondary structure according to amino acid hydropathy, (v) the statistical correlation of MW with protein secondary structures and their overall hydropathy, (vi) the statistical correlation of thymine in the second codon position with hydropathy and the energy cost of amino acid synthesis, and (vii) the statistical correlation of adenine in the second codon position with amino acid complexity and the MW of secondary protein structures. Amino acid physicochemical properties and functional constraints on proteins constitute a code that is translated into a purine bias within the coding DNA via tRNAs. In that sense, the Rrr pattern within coding DNA is the effect of information transfer on nucleotide composition from protein to DNA by selection according to the codon positions. Thus, coding DNA structure and ribosomal machinery co-evolved to minimize the energy cost of protein coding given the functional constraints on proteins.

RESUMEN

The role of glycosylation in the function of the T2 family of RNases is not well understood. In this work, we examined how glycosylation affects the progression of the T2 RNase Rny1p through the secretory pathway in Saccharomyces cerevisiae. We found that Rny1p requires entering into the ER first to become active and uses the adaptor protein Erv29p for packaging into COPII vesicles and transport to the Golgi apparatus. While inside the ER, Rny1p undergoes initial N-linked core glycosylation at four sites, N37, N70, N103 and N123. Rny1p transport to the Golgi results in the further attachment of high-glycans. Whereas modifications with glycans are dispensable for the nucleolytic activity of Rny1p, Golgi-mediated modifications are critical for its extracellular secretion. Failure of Golgi-specific glycosylation appears to direct Rny1p to the vacuole as an alternative destination and/or site of terminal degradation. These data reveal a previously unknown function of Golgi glycosylation in a T2 RNase as a sorting and secretion signal.

Asunto(s)

Aparato de Golgi/metabolismo , Ribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Exocitosis , Glicosilación , Polisacáridos/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Ribonucleasas/química , Ribonucleasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

In this study, we investigated the modalities of coding open reading frame (cORF) classification of expressed sequence tags (EST) by using the universal feature method (UFM). The UFM algorithm is based on the scoring of purine bias (Rrr) and stop codon frequencies. UFM classifies ORFs as coding or non-coding through a score based on 5 factors: (i) stop codon frequency; (ii) the product of the probabilities of purines occurring in the three positions of nucleotide triplets; (iii) the product of the probabilities of Cytosine (C), Guanine (G), and Adenine (A) occurring in the 1st, 2nd, and 3rd positions of triplets, respectively; (iv) the probabilities of a G occurring in the 1st and 2nd positions of triplets; and (v) the probabilities of a T occurring in the 1st and an A in the 2nd position of triplets. Because UFM is based on primary determinants of coding sequences that are conserved throughout the biosphere, it is suitable for cORF classification of any sequence in eukaryote transcriptomes without prior knowledge. Considering the protein sequences of the Protein Data Bank (RCSB PDB or more simply PDB) as a reference, we found that UFM classifies cORFs of ≥200 bp (if the coding strand is known) and cORFs of ≥300 bp (if the coding strand is unknown), and releases them in their coding strand and coding frame, which allows their automatic translation into protein sequences with a success rate equal to or higher than 95%. We first established the statistical parameters of UFM using ESTs from Plasmodium falciparum, Arabidopsis thaliana, Oryza sativa, Zea mays, Drosophila melanogaster, Homo sapiens and Chlamydomonas reinhardtii in reference to the protein sequences of PDB. Second, we showed that the success rate of cORF classification using UFM is expected to apply to approximately 95% of higher eukaryote genes that encode for proteins. Third, we used UFM in combination with CAP3 to assemble large EST samples into cORFs that we used to analyze transcriptome phenotypes in rice, maize, and humans. We discuss the error rate and the interference of noisy sequences such as pseudogenes, transposons, and retrotransposons. This method is suitable for rapid cORF extraction from transcriptome data and allows correct description of the genome phenotypes of plant genomes without prior knowledge. Additional care is necessary when addressing the human transcriptome due to the interference caused by large amounts of noisy sequences. UFM can be regarded as a low complexity tool for prior knowledge extraction concerning the coding fraction of the transcriptome of any eukaryote. Due to its low level of complexity, UFM is also very robust to variations of codon usage.