REGULACION POST-TRANSCRIPCIONAL MEDIADA POR LAS REGIONES 3' NO TRADUCIDAS DEL RNA MENSAJERO EN BACTERIAS

BFU2014-56698-P

Nombre agencia financiadora Ministerio de Economía y Competitividad
Acrónimo agencia financiadora MINECO
Programa Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia
Subprograma Subprograma Estatal de Generación del Conocimiento
Convocatoria Proyectos de I+D dentro del Subprograma Estatal de Generación del Conocimiento (2014)
Año convocatoria 2014
Unidad de gestión Dirección General de Investigación Científica y Técnica
Centro beneficiario AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS (CSIC)
Centro realización INSTITUTO DE AGROBIOTECNOLOGÍA Y RECURSOS NATURALES (IARN)
Identificador persistente http://dx.doi.org/10.13039/501100003329

Publicaciones

Found(s) 8 result(s)
Found(s) 1 page(s)

Un tránscrito antisentido conecta el estrés celular con la respuesta SOS

Digital.CSIC. Repositorio Institucional del CSIC
  • Bastet, Laurène
  • Caballero Sánchez, Carlos José
  • Villanueva, Maite
  • Lasa, Íñigo
  • Toledo-Arana, Alejandro
Póster presentado en la XI Reunión del Grupo de Microbiología Molecular de la Sociedad Española de Microbiología (SEM), celebrada en Sevilla del 6 al 8 de septiembre de 2016., La determinación precisa de los mapas transcriptómicos bacterianos ha revelado que el
número de regiones cromosómicas con transcripción solapante es mucho mayor de lo
que habíamos imaginado1. Estos RNAs antisentidos (asRNAs) se pueden generar de dos
maneras diferentes: i) por la presencia de un promotor situado en la cadena contraria a
una región codificante (CDS) ó ii) por la deficiencia en la parada de transcripción entre
genes convergentes. El segundo caso ocurre cuando no existe un terminador de
transcripción (TT) entre los genes o la estructura del TT es inestable, generando así
regiones 3’ solapantes1,2,3. También, existe la posibilidad de que la formación de la
estructura terminadora pueda ser modificada por factores externos tal y como ocurre en
algunos riboswitches. Todo ello hace que el nivel de antisentido dependa del promotor de
los genes convergentes y, si existe, del TT presente entre ellos. En cualquier caso, la
transcripción solapante en las regiones 3’, incrementa la complejidad de la organización
de los genes en el cromosoma al establecer interconexiones entre los mismos además de
la establecida por los operones. En un trabajo previo, descubrimos que esta transcripción
solapante afecta a la expresión de varios reguladores de Staphylococcus aureus. Uno de
ellos correspondía al gen lexA, que codifica para el principal regulador de la respuesta
SOS1. Mediante Northern blot determinamos que el transcrito antisentido se genera a
partir del promotor SigB del small RNA sbrB. Dicho transcrito incluye la CDS de SbrB, una
región que solapa completamente con el mRNA de lexA y la CDS de SosA. Para que este
RNA de 1,4 Kb se genere, la RNA polimerasa debe evitar el TT de sbrB, finalizando su
transcripción en el TT de sosA. El reemplazo del promotor SigB de sbrB por uno
constitutivo, provoca cambios en la expresión de LexA cuando la respuesta SOS es
inducida por Mitomicina, lo que demuestra la interconexión entre ambas vías de
regulación. Aunque son necesarios más experimentos para comprender este mecanismo
de regulación, este estudio ejemplifica la complejidad transcritómica presente en las
bacterias, donde un mismo transcrito puede contener múltiples elementos funcionales., Agradecimientos: al Ministerio de Economía y Competitividad y al Consejo Europeo de Investigación por la financiación de los proyectos de investigación BFU2014-56698-P y
ERC-2014-CoG-646869, respectivamente. A la Universidad Pública de Navarra por la financiación del contrato pre-doctoral de C.C., Peer Reviewed




Evolución diferencial de las 3’-UTRs en bacterias

Digital.CSIC. Repositorio Institucional del CSIC
  • Menéndez Gil, Pilar
  • Caballero Sánchez, Carlos José
  • Toledo-Arana, Alejandro
Póster presentado en la XI Reunión del Grupo de Microbiología Molecular de la Sociedad Española de Microbiología (SEM), celebrada en Sevilla del 6 al 8 de septiembre de 2016., Un RNA mensajero (mRNA) está formado por tres regiones bien diferenciadas, una región
central que codifica para la proteína correspondiente (CDS), la cual está rodeada por dos
regiones que no son traducidas y se denominan 5’-UTR (que comprende desde el inicio
de transcripción al inicio de traducción) y 3’-UTR (desde el final de la traducción al final de
la transcripción). En eucariotas, las 3’-UTRs son elementos esenciales para modular la
expresión de la proteína contenida en el mRNA. En esta región se encuentran los sitios
de reconocimiento para diversas proteínas de unión a RNA y las zonas de interacción con
los microRNAs que conjuntamente regulan la cantidad de proteína a traducir. Es
interesante destacar que la complejidad y longitud de las 3’-UTRs se relaciona de manera
directa con la evolución de los organismos. Es así que la longitud de las 3’-UTRs se
incrementa a medida que subimos en el árbol evolutivo. Es también llamativo que la
desregulación de ciertos genes causantes de algunas enfermedades como el cáncer está
asociado con el acortamiento de las 3’-UTRs. En bacterias, la función de estas regiones
es prácticamente desconocida. En trabajos previos de nuestro y otros grupos, se
demostró que las bacterias también contienen elementos reguladores en las 3’-UTRs para
modular la expresión proteica. Considerando que las 3’-UTRs han evolucionado de
manera diferente según los organismos, quisimos analizar si esto también ocurría en
procariotas. Así, basándonos en el mapa transcriptómico de S. aureus comparamos
genes con 3’-UTRs largas con sus respectivos homólogos en diferentes especies del
Género Staphylococcus. Sorprendentemente, descubrimos que la homología desaparecía
a la altura del codón de parada, lo que indicaba que cada gen homólogo tiene una 3’-UTR
diferente. Para comprobar si distintas 3’-UTRs provocan una expresión diferencial del
mismo gen homólogo, realizamos quimeras entre una CDS y las distintas 3’-UTRs y
medimos la expresión de la proteína. Los resultados mostraron que la expresión es
diferente según la 3’-UTR clonada. Esto sugiere que la regulación post-transcripcional
mediada por estas regiones ha evolucionado de manera diferente para un mismo gen
según en la especie bacteriana que se encuentre., Agradecimientos: al Ministerio de Economía y Competitividad y al Consejo Europeo de Investigación por la financiación de los proyectos de investigación BFU2014-56698-P y
ERC-2014-CoG-646869, respectivamente. A la Universidad Pública de Navarra por la financiación del contrato pre-doctoral de C.C., Peer Reviewed




The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Caballero Sánchez, Carlos José
  • Menéndez Gil, Pilar
  • Catalán Moreno, Arancha
  • Vergara-Irigaray, Marta
  • García, Begoña
  • Segura, Víctor
  • Irurzun, Naiara
  • Villanueva, Maite
  • Ruiz de los Mozos, Igor
  • Solano Goñi, Cristina
  • Lasa, Íñigo
  • Toledo-Arana, Alejandro
RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5′UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5′UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist., European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869]; Spanish Ministry of Economy and Competitiveness [BFU2011-23222, BIO2014-53530-R, BFU2014-56698-P]; Spanish National Research Council [CSIC-PII-201540I013]; C.J.C. was supported by predoctoral contract from the Public University of Navarre (UPNA), Spain. Funding for open access charge: European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme [646869]., Peer reviewed




A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Bronesky, Delphine
  • Desgranges, Emma
  • Corvaglia, Anna
  • François, Patrice
  • Caballero Sánchez, Carlos José
  • Prado, Laura
  • Toledo-Arana, Alejandro
  • Lasa, Íñigo
  • Moreau, Karan
  • Vandenesch, François
  • Marzi, Stefano
  • Romby, Pascale
  • Caldelari, Isabelle
Pathogenic bacteria must rapidly adapt to ever‐changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA‐repressed small non‐coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3′ untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose‐6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested., This work was supported by the Centre National de la Recherche Scientifique (CNRS) to [P.R.] and by the Agence Nationale de la Recherche (ANR, grant ANR‐16‐CE11‐0007‐01, RIBOSTAPH, to [P.R.]). It has also been published under the framework of the LABEX: ANR‐10‐LABX‐0036 NETRNA to [P.R.], a funding from the state managed by the French National Research Agency as part of the investments for the future program. The work is financed by a “Projet international de coopération scientifique” (PICS) No. PICS07507 between France and Spain to [I.C.]. D. Bronesky was supported by Fondation pour la Recherche Médicale (FDT20160435025). A. T‐A is financed by the Spanish Ministry of Economy and Competitiveness (BFU2014‐56698‐P) and the European Research Council Consolidator Grant (646869‐ReguloBac‐3UTR)., Peer reviewed




Differential evolution in 3′UTRs leads to specific gene expression in Staphylococcus

Digital.CSIC. Repositorio Institucional del CSIC
  • Menéndez Gil, Pilar
  • Caballero Sánchez, Carlos José
  • Catalán Moreno, Arancha
  • Irurzun, Naiara
  • Barrio-Hernandez, Iñigo
  • Caldelari, Isabelle
  • Toledo-Arana, Alejandro
The evolution of gene expression regulation has contributed to species differentiation. The 3′ untranslated regions (3′UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3′UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3′UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3′UTR of orthologous genes and demonstrated that 3′UTR sequence variations affect protein production. This suggested that species-specific functional 3′UTRs might be specifically selected during evolution. 3′UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3′UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3′UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3′UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity., A.T.-A. was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [ERC-CoG-2014-646869] and the Spanish Ministry of Economy and Competitiveness [BFU2014-56698-P] grants; C.J.C. was supported by a predoctoral contract from the Public University of Navarre; I.C. was supported by recurrent funding from the Centre National de la Recherche Scientifique (CNRS) and by a PICS program (‘Projet International de Coopération Scientifique’, No. PICS07507, CNRS) between France and Spain. Funding for open access charge: European Research Council (ERC), European Union's Horizon 2020 research and innovation program [ERC-CoG-2014-646869].




One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Catalán Moreno, Arancha
  • Caballero Sánchez, Carlos José
  • Irurzun, Naiara
  • Cuesta, Sergio
  • Lopez-Sagaseta, Jacinto
  • Toledo-Arana, Alejandro
Bacterial genomes encode several families of protein paralogs. Discrimination between functional divergence and redundancy among paralogs is challenging due to their sequence conservation. Here, we investigated whether the amino acid differences present in the cold shock protein (CSP) paralogs of Staphylococcus aureus were responsible for functional specificity. Since deletion of cspA reduces the synthesis of staphyloxanthin (STX), we used it as an in vivo reporter of CSP functionality. Complementation of a ΔcspA strain with the different S. aureus CSP variants showed that only CspA could specifically restore STX production by controlling the activity of the stress-associated sigma B factor (σ). To determine the amino acid residues responsible for CspA specificity, we created several chimeric CSPs that interchanged the amino acid differences between CspA and CspC, which shared the highest identity. We demonstrated that CspA Pro58 was responsible for the specific control of σ activity and its associated phenotypes. Interestingly, CspC gained the biological function of CspA when the E58P substitution was introduced. This study highlights how just one evolutionarily selected amino acid change may be sufficient to modify the specific functionality of CSP paralogs., his work was supported by the European Research Council (ERC), under the European Union Horizon 2020 research and innovation program (Grant Agreement No. 646869), and the Spanish Ministry of Economy and Competitiveness (BFU2014‐56698‐P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre, Spain. J.L.‐S. is a recipient of a Ramón y Cajal Contract (RYC‐2017‐21683).




Fluorescent molecular beacons mimicking RNA secondary structures to study RNA chaperone activity

Digital.CSIC. Repositorio Institucional del CSIC
  • Menéndez Gil, Pilar
  • Caballero Sánchez, Carlos José
  • Solano Goñi, Cristina
  • Toledo-Arana, Alejandro
Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases., This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Grant Agreement No. 646869) and the Spanish Ministry of Economy and Competitiveness (BFU2014-56698-P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre (UPNA), Spain.




The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus

Dadun. Depósito Académico Digital de la Universidad de Navarra
  • Caballero, C.J. (Carlos J.)
  • Menendez-Gil, P. (Pilar)
  • Catalan-Moreno, A. (Arancha)
  • Vergara-Irigaray, M. (Marta)
  • García, B. (Begoña)
  • Segura, V. (Víctor)
  • Irurzun, N. (Naiara)
  • Villanueva, M. (Maite)
  • Ruiz-de-los-Mozos, I. (Igor)
  • Solano, C. (Cristina)
  • Lasa, I. (Íñigo)
RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.