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Instituto de Parasitología y Biomedicina
"López - Neyra"
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[ Staff | Summary of Research | Funding Agencies | Publications | Doctoral Theses | Patents | Teaching]




BIOLOGICAL ACTIVITY OF THE RNA



Group Leader
    • Alfredo Berzal Herranz     
        email: aberzalh (@ipb.csic.es)
        Tlf: 958181648



    Staff Research Posdoctoral
    • Cristina Romero López     


    Staff Research Predoctoral
    • María Isabel Pérez Jover     


    Technical Assistants
    • Beatriz Berzal Herranz     


    Authorized Staff
    • Marina Quesada Martínez     

     

    SUMMARY OF RESEARCH


     

    STRUCTURE/FUNCTION OF CONSERVED GENOMIC RNA DOMAINS IN RNA VIRUS.
    The research group is co-leaded by Alfredo Berzal-Herranz and Cristina Romero-López.

     

    The lack of knowledge about the functions performed by RNA in living organisms has made that RNA has been long underrated as simply player in the transmission of the genetic information from DNA to proteins. The identification of new classes of RNAs, grouped under the name of non-coding RNAs (ncRNAs), and the elucidation of the functions they perform have placed RNA at the center of biological processes essential for life. The ncRNAs, exert a function by themselves, are functional RNAs, demonstrating the existence of functional genetic information beyond the one that encodes proteins. Their study has also revealed the existence of an additional system of regulation of gene expression that involves different types of non-coding RNAs. The accumulation of information on biological activity of RNA molecules has allowed establishing the molecular basis of different pathological processes, as well as to determine that errors in the RNA metabolism, or the loss of function of RNA molecules are the cause of numerous diseases or pathologies of great sanitary importance.


    Viral RNA genomes constitute a particular type of functional RNAs. They are compact genomes that contain all the required information for the completion of the viral infective cycle. To achieve this in a small genome, RNA viruses have developed a system for storing and encoding genetic information beyond the nucleotide sequence that is independent and complementary to the information that encodes proteins. The information is stored in the form of discrete structural elements responsible for essential functions. These structural elements are highly conserved, and distributed throughout the viral genome, despite the existence of genomic regions with a greater concentration of functional RNA elements have been defined, for example non-translatable regions or UTRs. RNA elements exert their function by establishing long-distance RNA-RNA interactions between them and / or by recruiting cellular or viral factors. Thus their functionality is achieved by the establishment of a complex global structure specific of any of the essential functions, e.g., replication, translation, encapsidation.


    The hepatitis C virus (HCV) genome is an approximately 9,600 nt-long positive, single-stranded RNA molecule that codes for a single open reading frame flanked by untranslated regions (UTRs). The UTRs are highly conserved in sequence and structure between the different viral isolates, which contrasts with the great genetic variability of the viral genome. In early infection, HCV protein synthesis is initiated by an internal ribosome entry site (IRES)-dependent mechanism different to the cap-dependent method used for the translation of most cellular mRNAs. The IRES element is mostly located at the 5'UTR and spans a short stretch of the core coding sequence. It folds into a well conserved compact structure that contains a subset of well characterized domains and subdomains. These structural elements guide the direct recruitment of the 40S ribosomal subunit and the further binding of eIF3 which aids the incorporation of the ternary complex eIF2-tRNAi Met and the joining of the 60S subunit. This mechanism minimizes protein factor requirements and simplifies the pathway for the assembly of the translationally active 80S ribosome. Further, the presence of domains at the 3' end of the genomic RNA influences translation efficiency, most likely by the acquisition of a closed-loop topology resembling the circular structure adopted by cellular mRNAs. Similarly, the 3'UTR contains several highly conserved functional RNA domains required for replication, translation and viral genome dimerization.


    In addition to RNA domains being located in the untranslated regions, numerous cis-acting signals have been identified in the ORFs of RNA viruses. These RNA elements are bifunctional, they simultaneously hold protein coding information and perform specific functions. Thus in the HCV RNA genome a conserved structural region within the 3' end of the viral polymerase (NS5B) coding sequence termed as CRE (cis-acting replication element) has been defined as essential for viral replication. CRE is composed of three stem-loops, being the central one, 5BSL3.2, indispensable for HCV propagation. The 5BSL3.2 is a 48 nt-long RNA domain that folds into a stable stem closed by a 12 nt-long loop. The stem is interrupted by an 8 nt-long bulge, which we described that is involved in a long distance RNA/RNA interaction with the apical loop of the IRES' subdomain IIId at the 5'UTR. This interaction that takes place in the absence of proteins we also demonstrated that is involved in the regulation of the IRES activity. Further studies have showed the existence of a complex network of RNA/RNA interactions involving at least the IRES, the 3'UTR and the CRE regions. Our results have allowed proposing that the 5BSL3.2 domain plays a central role in this network of interactions regulating the switching between the different stages of the viral cycle.


    HCV is the prototype of the genus Hepacivirus a member of the family Flaviviridae. The largest genus of this family is the genus Flavivirus, which includes important human pathogens like dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), yellow fever (YFV), among others. The flaviviral genome consists on a positive-sense single-stranded RNA molecule approximately 11,000 nt-long, varying depending on the species. It bears a type 1 cap structure at its 50 end (m7GpppAmp) but it lacks a polyA tail in the 3' end. The RNA genome contains a single ORF flanked by untranslated regions (UTRs). It serves as a messenger for the synthesis of a single polyprotein that is processed by viral and cellular proteases to yield 10 different products. The flanking UTRs are defined by discrete, functionally active structural RNA elements that play important roles in the viral cycle.


    An efficient strategy to deepen the characterization of the functional RNA domains is the use of nucleic acids as tools. In particular RNA molecules have shown to efficiently interfere with the structure of RNA motifs and to compete out RNA/RNA interactions, therefore to interfere with the functionality of RNA domains. Among these tools stand out for their great potential the aptamers. Aptamers are RNA or DNA oligonucleotides that specifically and efficiently bind to a ligand molecule. We have isolated collections of aptamers selected against RNA domains of the HCV and WNV genomes. Their characterization has allowed us identifying genomic sequences and structural domains as potential antiviral targets.


    Our research line is the study or the structure/function of viral RNA domains. Currently we are focused in the characterization of these domains in the genomes of the HCV and WNV. We also want to explore its potential as antiviral or therapeutic targets. In parallel, we address the development of general-purpose strategies for the design and application of RNA molecules as efficient tools in biotechnology and basic and biomedical research.



     


    FUNDING AGENCIES LAST 5 YEARS

    - FALTA JUSTIFICANTE DE FIRMA ELECTRONICA - ENVIAR A PROYECTOS.NACIONALES@CSIC.ES - ESTRUCTURA/FUNCION DE DOMINIOS RNA CONSERVADOS EN EL GENOMA DEL VIRUS DE LA HEPATITIS C (HCV). PROYECTO, PN2015 - PROY I+D - S. E. G. C. - P. EXCELENCIA, Ref: BFU2015-64359-P, (2016 - 2018).

    - Estudio del fenómeno de circularizacion del RNA genómico del virus de la hepatitis C y su potencial uso como diana terapéutica. PROYECTO, , Ref: P11-CVI-7430, (2013 - 2016).

    - Aptámeros RNA. Herramientas moleculares para la caracterización funcional del dominio RNA genómico CRE del virus de la hepatitis C. PROYECTO, , Ref: BFU2012-31213, (2013 - 2015).

    - RED Nacional RNA: Estructura, función y Aplicaciones Biomédicas y Biotecnológicas - RIBORED. ACCION ESPECIAL, INV.FUND.NO ORIENTADA.- BIOLOGIA FUNDAMENTAL, Ref: BFU2009-07883-E, (2010 - 2014).

    - Identificación y optimización de RNAs que interfieren específicamente la replicación del VHC. RNA como base para el desarrollo de herramientas antivirales. PROYECTO, PLAN ANDALUZ DE INVESTIGACION, Ref: P09-CTS-5077, (2010 - 2013).

    - Identificación de señales en genomas virales RNA. PROYECTO, PROYECTOS INTRAMURALES, Ref: 201120E004, (2010 - 2014).

    - Caracterización de motivos RNA funcionales de estructura conservada en genomas RNA. Uso de aptameros RNA como herramientas moleculares. PROYECTO, INV.FUND.NO ORIENTADA.- BIOLOGIA FUNDAMENTAL, Ref: BFU2009-08137, (2010 - 2014).

     

     

    PUBLICATIONS LAST 5 YEARS

    -Romero-López, C.; Barroso-Deljesus, A.; Berzal-Herranz, A., The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization, Scientific Reports, 2017, Vol. 7: 43415, ARTICULO, Id:1540

    -Romero-López, C.; Berzal-Herranz, A., The 5BSL3.2 functional RNA domain connects distant regions in the hepatitis C virus genome, Frontiers in Microbiology, 2017, Vol. 8: 2093, ARTICULO DE REVISION, Id:1539

    -Fernández-Sanlés, A.; Ríos-Marco, P.; Romero-López, C.; Berzal-Herranz, A., Functional information stored in the conserved structural RNA domains of flavivirus genomes, Frontiers in Microbiology, 2017, Vol. 8: 546, ARTICULO DE REVISION, Id:1510

    -Romero-López, C.; Lahlali, T.; Berzal-Herranz, B.; Berzal-Herranz, A., Development of optimized inhibitor RNAs allowing multisite-targeting of the HCV genome, Molecules, 2017, Vol. 22: 5-861, ARTICULO, Id:1501

    -Romero-López, C.; Berzal-Herranz, A., Aptamers: Biomedical interest and applications, Pharmaceuticals, 2017, Vol. 10: 1-32, EDITORIAL, Id:1496

    -Pablo Ríos-Marco; Cristina Romero-López; Alfredo Berzal-Herranz, The essential cis-acting replication element of the HCV genome recruits regulatory host factors that influence viral replication and translation, Scientific Reports, 2016, Vol. 6: 25729, ARTICULO, Id:1375

    -Torrecilla, J.; del Pozo-Rodríguez, A.; Solinis, M.A.; Apaolaza, P.S.; Berzal-Herranz, B.; Romero-López, C.; Berzal-Herranz, A.; Rodríguez-Gascón, A., Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES)., Colloids and Surfaces B: Biointerfaces, 2016, Vol. 146: 808-817, ARTICULO, Id:1368

    -Lahlali, T.; Plissonnier, M.L.; Romero-López, C.; Michelet, M.; Ducarouge, B.; Berzal-Herranz, A.; Zoulim, F.; Mehlen, P.; Parent, R., Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response, CMGH Cellular and Molecular Gastroenterology and Hepatology, 2016, Vol. 2: 281-301.e9, ARTICULO, Id:1356

    -López-Aguilar, C.; Romero-López, C.; Espinosa, M.; Berzal-Herranz, A.; del Solar, G., The 5'-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB, Front. Genet., 2015, Vol. 6: JUN-225, ARTICULO, Id:1469

    -Fernández-Sanlés, A.; Berzal-Herranz, B.; González-Matamala, R.; Ríos-Marco, P.; Romero-López, C.; Berzal-Herranz, A., RNA aptamers as molecular tools to study the functionality of the Hepatitis C Virus CRE region, Molecules, 2015, Vol. 20: 16030-16047, ARTICULO, Id:1460

    -Reyes-Darias, J.A.; Sánchez-Luque, F.J.; Morales, J.C.; Pérez-Rentero, S.; Eritja, R.; Berzal-Herranz, A., Glucose conjugation of anti-HIV-1 oligonucleotides containing unmethylated CpG motifs reduces their immunostimulatory activity, Chembiochem : a European journal of chemical biology, 2015, Vol. 16: 584-591, ARTICULO, Id:1424

    -Romero-López C.; Berzal-Herranz, A., Current and emerging themes in the structural analysis of viral RNA genomes. Applications for the development of novel therapeutic drugs, Genomics and Computacional Biology, 2015, Vol. 1: 1-e15, ARTICULO DE REVISION, Id:1407

    -Romero-Lopez, C.; Barroso-DelJesus, A.; Garcia-Sacristán, A.; Briones, C.; Berzal-Herranz, A., End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 30X-tail region, Nucleic Acids Research, 2014, Vol. 42: 567-582, ARTÍCULO, Id:1193

    -Sánchez-Luque, F.J; Stich, M; Manrubia, S; Briones, C; Berzal-Herranz, A., Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimization strategies, Scientific Reports, 2014, Vol. 4: 6242, ARTÍCULO, Id:1192

    -Robaldo, L; Berzal-Herranz, A; Montserrat, J.M; Iribarren, A.M, Activity of core-modified 10-23 DNAzymes against HCV, ChemMedChem, 2014, Vol. 9: 2172-2177, ARTÍCULO, Id:1164

    -Romero-López C; Berzal-Herranz, A, Structure-function relationship in viral RNA genomes. The case of HCV, World Journal of Medical Genetics, 2014, Vol. 4: 6-18, ARTICULO DE REVISION, Id:1160

    -Reyes-Darias, J.A.; Berzal-Herranz, A., Detection of immune response activation by exogenous nucleic acids by a multiplex RT-PCR method, Molecular and Cellular Probes, 2014, Vol. 28: 181-185, ARTÍCULO, Id:1159

    -Romero-López, C.; Berzal-Herranz, A., Unmasking the information encoded as structural motifs of viral RNA genomes: A potential antiviral target, Reviews in Medical Virology, 2013, Vol. 23: 340-354, ARTICULO DE REVISION, Id:1061

    -Marton, S.; Romero-López, C.; Berzal-Herranz, A., RNA aptamer-mediated interference of HCV replication by targeting the CRE-5BSL3.2 domain, Journal of Viral Hepatitis, 2013, Vol. 20: 103-112, ARTÍCULO, Id:1045


     

     

    DOCTORAL THESES LAST 5 YEARS

     

    2013

    Francisco José Sánchez Luque

    RNAs Inhibidores frente al Virus de la Inmunodeficiencia Humana

    Instituto de Parasitología y Biomedicina "López-Neyra" CSIC-Universidad de Granada

     

     

     

    PATENTS LAST 5 YEARS

     

    - MOLECULAS INHIBIDORAS DEL VIRUS DE LA INMUNODEFICIENCIA HUMANA TIPO 1 (VIH-1), PROCEDIMIENTO DE OBTENCION Y SUS APLICACIONES. PCT/ES13/070809, 2013

     


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