Computational design and designability of gene regulatory networks

Nuestro conocimiento de las interacciones moleculares nos ha conducido hoy hacia una perspectiva ingenieril, donde diseños e implementaciones de sistemas artificiales de regulación intentan proporcionar instrucciones fundamentales para la reprogramación celular. Nosotros aquí abordamos el diseño de redes de genes como una forma de profundizar en la comprensión de las regulaciones naturales. También abordamos el problema de la diseñabilidad dada una genoteca de elementos compatibles. Con este fin, aplicamos métodos heuríticos de optimización que implementan rutinas para resolver problemas inversos, así como herramientas de análisis matemático para estudiar la dinámica de la expresión genética. Debido a que la ingeniería de redes de transcripción se ha basado principalmente en el ensamblaje de unos pocos elementos regulatorios usando principios de diseño racional, desarrollamos un marco de diseño computacional para explotar este enfoque. Modelos asociados a genotecas fueron examinados para descubrir el espacio genotípico asociado a un cierto fenotipo. Además, desarrollamos un procedimiento completamente automatizado para diseñar moleculas de ARN no codificante con capacidad regulatoria, basándonos en un modelo fisicoquímico y aprovechando la regulación alostérica. Los circuitos de ARN resultantes implementaban un mecanismo de control post-transcripcional para la expresión de proteínas que podía ser combinado con elementos transcripcionales. También aplicamos los métodos heurísticos para analizar la diseñabilidad de rutas metabólicas. Ciertamente, los métodos de diseño computacional pueden al mismo tiempo aprender de los mecanismos naturales con el fin de explotar sus principios fundamentales. Así, los estudios de estos sistemas nos permiten profundizar en la ingeniería genética. De relevancia, el control integral y las regulaciones incoherentes son estrategias generales que los organismos emplean y que aquí analizamos.Rodrigo Tarrega, G. (2011). Computational design and designability of gene regulatory networks [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1417

Virus-host interactome: Putting the accent on how it changes

[EN] Viral infections are extremely complex processes that could only be well understood by precisely characterizing the interaction networks between the virus and the host components. In recent years, much effort has gone in this directionwith the aimof unveiling themolecular basis of viral pathology. These networks are mostly formed by viral and host proteins, and are expected to be dynamic bothwith time and space (i.e., with the progression of infection, as well as with the virus and host genotypes; what we call plastodynamic). This largely overlooked spatio-temporal evolution urgently calls for a change both in the conceptual paradigms and experimental techniques used so far to characterize virus-host interactions. More generally, molecular plasticity and temporal dynamics are unavoidable components of themechanisms that underlie any complex disease; components whose understandingwill eventually enhance our ability to modulate those networkswith the aimof improving disease treatments.This work is supported by the grants BFU2015-66894-P (to G.R.), BI02014-54269-R (to J-A.D.) and BFU2015-65037-P (to S.F.E.) from the Ministerio de Economia, Industria y Competitividad, and by the grant PROMETEOII/2014/021 from the Generalitat Valenciana (to S.F.E. and J-A.D.).Rodrigo Tarrega, G.; Daros Arnau, JA.; Elena Fito, SF. (2017). Virus-host interactome: Putting the accent on how it changes. Journal of Proteomics. 156:1-4. https://doi.org/10.1016/j.jprot.2016.12.007S1415

Viral Fitness Correlates with the Magnitude and Direction o the Perturbation Induced in the Host's Transcriptome: The Tobacco Etch Potyvirus-Tobacco Case Study

[EN] Determining the fitness of viral genotypes has become a standard practice in virology as it is essential to evaluate their evolutionary potential. Darwinian fitness, defined as the advantage of a given genotype with respect to a reference one, is a complex property that captures, in a single figure, differences in performance at every stage of viral infection. To what extent does viral fitness result from specific molecular interactions with host factors and regulatory networks during infection? Can we identify host genes in functional classes whose expression depends on viral fitness? Here, we compared the transcriptomes of tobacco plants infected with seven genotypes of tobacco etch potyvirus that differ in fitness. We found that the larger the fitness differences among genotypes, the more dissimilar the transcriptomic profiles are. Consistently, two different mutations, one in the viral RNA polymerase and another in the viral suppressor of RNA silencing, resulted in significantly similar gene expression profiles. Moreover, we identified host genes whose expression showed a significant correlation, positive or negative, with the virus' fitness. Differentially expressed genes which were positively correlated with viral fitness activate hormone- and RNA silencing-mediated pathways of plant defense. In contrast, those that were negatively correlated with fitness affect metabolism, reducing growth, and development. Overall, these results reveal the high information content of viral fitness and suggest its potential use to predict differences in genomic profiles of infected hosts.We thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance, the EvolSysVir lab members for help, comments and discussions, Rachel Whitaker for English proofreading, and Lorena Latorre (IBMCP Genomics Service) and Javier Forment (IBMCP Bioinformatics Service) for their assistance. This research was supported by grants from Spain's Agencia Estatal de Investigacion-FEDER (BFU2012-30805 and BFU2015-65037-P to S.F.E. and BFU2015-66894-P to G.R.) and Generalitat Valenciana (PROMETEOII/2014/021).Cervera-Benet, H.; Ambros Palaguerri, S.; Bernet, GP.; Rodrigo Tarrega, G.; Elena Fito, SF. (2018). Viral Fitness Correlates with the Magnitude and Direction o the Perturbation Induced in the Host's Transcriptome: The Tobacco Etch Potyvirus-Tobacco Case Study. 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Transcriptomic changes in Nicotiana benthamiana plants inoculated with the wild-type or an attenuated mutant of Tobacco vein banding mosaic virus. Molecular Plant Pathology, 18(8), 1175-1188. doi:10.1111/mpp.12471Hafrén, A., Lõhmus, A., & Mäkinen, K. (2015). Formation of Potato Virus A-Induced RNA Granules and Viral Translation Are Interrelated Processes Required for Optimal Virus Accumulation. PLOS Pathogens, 11(12), e1005314. doi:10.1371/journal.ppat.1005314Henty-Ridilla, J. L., Li, J., Day, B., & Staiger, C. J. (2014). ACTIN DEPOLYMERIZING FACTOR4 Regulates Actin Dynamics during Innate Immune Signaling in Arabidopsis. The Plant Cell, 26(1), 340-352. doi:10.1105/tpc.113.122499Hillung, J., Cuevas, J. M., Valverde, S., & Elena, S. F. (2014). EXPERIMENTAL EVOLUTION OF AN EMERGING PLANT VIRUS IN HOST GENOTYPES THAT DIFFER IN THEIR SUSCEPTIBILITY TO INFECTION. Evolution, 68(9), 2467-2480. doi:10.1111/evo.12458Hillung, J., García-García, F., Dopazo, J., Cuevas, J. M., & Elena, S. F. (2016). The transcriptomics of an experimentally evolved plant-virus interaction. Scientific Reports, 6(1). doi:10.1038/srep24901Hyun, T. K., Albacete, A., van der Graaff, E., Eom, S. H., Großkinsky, D. K., Böhm, H., … Roitsch, T. (2015). The Arabidopsis PLAT domain protein1 promotes abiotic stress tolerance and growth in tobacco. Transgenic Research, 24(4), 651-663. doi:10.1007/s11248-015-9868-6Lalić, J., Agudelo-Romero, P., Carrasco, P., & Elena, S. F. (2010). Adaptation of tobacco etch potyvirus to a susceptible ecotype of Arabidopsis thaliana capacitates it for systemic infection of resistant ecotypes. Philosophical Transactions of the Royal Society B: Biological Sciences, 365(1548), 1997-2007. doi:10.1098/rstb.2010.0044Lalić, J., Cuevas, J. M., & Elena, S. F. (2011). Effect of Host Species on the Distribution of Mutational Fitness Effects for an RNA Virus. PLoS Genetics, 7(11), e1002378. doi:10.1371/journal.pgen.1002378Lalić, J., & Elena, S. F. (2013). Epistasis between mutations is host-dependent for an RNA virus. Biology Letters, 9(1), 20120396. doi:10.1098/rsbl.2012.0396Lalić, J., & Elena, S. F. (2015). The impact of high-order epistasis in the within-host fitness of a positive-sense plant RNA virus. Journal of Evolutionary Biology, 28(12), 2236-2247. doi:10.1111/jeb.12748Lamesch, P., Berardini, T. Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., … Huala, E. (2011). The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Research, 40(D1), D1202-D1210. doi:10.1093/nar/gkr1090Li, Y., Jing, Y., Li, J., Xu, G., & Lin, R. (2014). Arabidopsis VQ MOTIF-CONTAINING PROTEIN29 Represses Seedling Deetiolation by Interacting with PHYTOCHROME-INTERACTING FACTOR1. Plant Physiology, 164(4), 2068-2080. doi:10.1104/pp.113.234492Linnen, C. R., & Hoekstra, H. E. (2009). Measuring Natural Selection on Genotypes and Phenotypes in the Wild. 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Genome rearrangement affects RNA virus adaptability on prostate cancer cells. Frontiers in Genetics, 6. doi:10.3389/fgene.2015.00121Revers, F., & García, J. A. (2015). Molecular Biology of Potyviruses. Advances in Virus Research, 101-199. doi:10.1016/bs.aivir.2014.11.006Robaglia, C., & Caranta, C. (2006). Translation initiation factors: a weak link in plant RNA virus infection. Trends in Plant Science, 11(1), 40-45. doi:10.1016/j.tplants.2005.11.004Rodrigo, G., Carrera, J., Ruiz-Ferrer, V., del Toro, F. J., Llave, C., Voinnet, O., & Elena, S. F. (2012). A Meta-Analysis Reveals the Commonalities and Differences in Arabidopsis thaliana Response to Different Viral Pathogens. PLoS ONE, 7(7), e40526. doi:10.1371/journal.pone.0040526Sánchez, F., Manrique, P., Mansilla, C., Lunello, P., Wang, X., Rodrigo, G., … Ponz, F. (2015). Viral Strain-Specific Differential Alterations in Arabidopsis Developmental Patterns. 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THE DISTRIBUTION OF MUTATIONAL FITNESS EFFECTS OF PHAGE φX174 ON DIFFERENT HOSTS. Evolution, 66(11), 3495-3507. doi:10.1111/j.1558-5646.2012.01691.xVijayapalani, P., Maeshima, M., Nagasaki-Takekuchi, N., & Miller, W. A. (2012). Interaction of the Trans-Frame Potyvirus Protein P3N-PIPO with Host Protein PCaP1 Facilitates Potyvirus Movement. PLoS Pathogens, 8(4), e1002639. doi:10.1371/journal.ppat.1002639Visher, E., Whitefield, S. E., McCrone, J. T., Fitzsimmons, W., & Lauring, A. S. (2016). The Mutational Robustness of Influenza A Virus. PLOS Pathogens, 12(8), e1005856. doi:10.1371/journal.ppat.1005856Viswanathan, K., & Früh, K. (2007). Viral proteomics: global evaluation of viruses and their interaction with the host. Expert Review of Proteomics, 4(6), 815-829. doi:10.1586/14789450.4.6.815Wachter, A., & Hartmann, L. (2014). NMD: Nonsense-Mediated Defense. Cell Host & Microbe, 16(3), 273-275. doi:10.1016/j.chom.2014.08.015Wargo, A. R., & Kurath, G. (2012). 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CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA

[EN] DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of substrates and reactions (hardware) for DNA computing.We thank V. Aragones (IBMCP) for her technical assistance on PAGE. The work was supported by the Spanish Ministry of Economy and Competitiveness grants BFU2015-66894-P (to GR) and BIO2017-83184-R (to JAD) and by the Spanish Ministry of Science, Innovation, and Universities grant PGC2018-101410-B-I00 (to GR); grants cofinanced by the European Regional Development Fund.Montagud-Martínez, R.; Heras-Hernández, M.; Goiriz, L.; Rodrigo Tarrega, G.; Daròs, J. (2021). CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA. ACS Synthetic Biology. 10(5):950-956. https://doi.org/10.1021/acssynbio.0c0064995095610

A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes

[EN] Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein-protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators. We verified a subset of 25 interactions in planta by bimolecular fluorescence complementation assays. We then constructed and analyzed a network comprising 399 TuMV-A. thaliana interactions together with intravirus and intrahost connections. In particular, we found that the host proteins targeted by TuMV are enriched in different aspects of plant responses to infections, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. The proviral or antiviral role of ten host proteins was validated by characterizing the infection dynamics in the corresponding mutant plants, supporting a proviral role for the transcriptional regulator TGA1. Comparison with similar studies with animal viruses, highlights shared fundamental features in their mode of action.We thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance and the rest of the EvolSysVir lab for fruitful discussions. This work was supported by grants PID2019-103998GB-I00 and PGC2018-101410-B-I00 (Agencia Estatal de Investigacion - FEDER) to S.F.E. and G.R., respectively, and PROMETEO/2019/012 (Generalitat Valenciana) to S.F.E.Martínez, F.; Carrasco, JL.; Toft, C.; Hillung, J.; Giménez-Santamarina, S.; Yenush, L.; Rodrigo Tarrega, G.... (2023). A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes. Communications Biology. 6(1):1-18. https://doi.org/10.1038/s42003-023-04427-81186

Molecular signatures of silencing suppression degeneracy from a complex RNA virus

[EN] As genomic architectures become more complex, they begin to accumulate degenerate and redundant elements. However, analyses of the molecular mechanisms underlying these genetic architecture features remain scarce, especially in compact but sufficiently complex genomes. In the present study, we followed a proteomic approach together with a computational network analysis to reveal molecular signatures of protein function degeneracy from a plant virus (as virus-host protein-protein interactions). We employed affinity purification coupled to mass spectrometry to detect several host factors interacting with two proteins of Citrus tristeza virus (p20 and p25) that are known to function as RNA silencing suppressors, using an experimental system of transient expression in a model plant. The study was expanded by considering two different isolates of the virus, and some key interactions were confirmed by bimolecular fluorescence complementation assays. We found that p20 and p25 target a common set of plant proteins including chloroplastic proteins and translation factors. Moreover, we noted that even specific targets of each viral protein overlap in function. Notably, we identified argonaute proteins (key players in RNA silencing) as reliable targets of p20. Furthermore, we found that these viral proteins preferentially do not target hubs in the host protein interactome, but elements that can transfer information by bridging different parts of the interactome. Overall, our results demonstrate that two distinct proteins encoded in the same viral genome that overlap in function also overlap in their interactions with the cell proteome, thereby highlighting an overlooked connection from a degenerate viral system.This work was supported by the grants PROMETEO/2019/012 from the Valencian Regional Government (to SA), as well as grants AGL2010-20221/AGR, BIO2017-83184-R, and PGC2018-101410-B-I00 from the Spanish Ministry of Science, Innovation, and Universities (to SA, JAD, and GR, respectively). The two latter grants were co-financed by the European Regional Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ambrós, S.; Gómez-Muñoz, N.; Giménez-Santamarina, S.; Sánchez-Vicente, J.; Navarro-López, J.; Martínez, F.; Daròs, J.... (2021). Molecular signatures of silencing suppression degeneracy from a complex RNA virus. PLoS Computational Biology. 17(6):1-21. https://doi.org/10.1371/journal.pcbi.1009166S12117

Análisis y diseño de circuitos genéticos sintéticos para aplicaciones energéticas y para la participación del equipo valenciano en la competición "International Genetically Engineered Machine" organizada por el Massachusetts Institute of Technology

Proyecto ConfidencialRodrigo Tárrega, GJ. (2006). Análisis y diseño de circuitos genéticos sintéticos para aplicaciones energéticas y para la participación del equipo valenciano en la competición "International Genetically Engineered Machine" organizada por el Massachusetts Institute of Technology. http://hdl.handle.net/10251/36895.Archivo delegad

CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA

[EN] DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of substrates and reactions (hardware) for DNA computing.We thank V. Aragones (IBMCP) for her technical assistance on PAGE. The work was supported by the Spanish Ministry of Economy and Competitiveness grants BFU2015-66894-P (to GR) and BIO2017-83184-R (to JAD) and by the Spanish Ministry of Science, Innovation, and Universities grant PGC2018-101410-B-I00 (to GR); grants cofinanced by the European Regional Development Fund.Montagud-Martínez, R.; Heras-Hernández, M.; Goiriz, L.; Rodrigo Tarrega, G.; Daròs, J. (2021). CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA. ACS Synthetic Biology. 10(5):950-956. https://doi.org/10.1021/acssynbio.0c0064995095610

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation

[EN] The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.We thank M. Carmen Montaner, Javier Peman, Miguel Salavert, and Alba Ruiz from the Hospital Universitario y Politecnico La Fe for their support in the collection of the fecal samples. This work was supported by the Fondo Supera COVID-19 from CRUE and Banco Santander (Grant COV-CRISPIS, BOE-A-2020-7995, to GR), the CSIC PTI Salud Global (Grant SGL2021-03-040, to GR, and Grants 202020E292 and SGL2103034, to PDC) through the European Union-Next Generation EU, the Fondo COVID-19 from the Instituto de Salud Carlos III (Grant COV20/00210, to PDC), and the Generalitat Valenciana (Grant SEJI/2020/011, to GR). PDC was supported by the Ramon y Cajal program (RYC2019-028015-I) and RMC by a predoctoral fellowship (PRE2019-088531), both from the Spanish Ministerio de Ciencia e Innovacion.Marqués, M.; Ruiz, R.; Montagud-Martínez, R.; Márquez-Costa, R.; Albert, S.; Domingo-Calap, P.; Rodrigo Tarrega, G. (2021). CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation. ACS Synthetic Biology. 10(12):3595-3599. https://doi.org/10.1021/acssynbio.1c00323S35953599101