Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants

ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO–sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO–sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO–RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing.This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C.Carbonell Olivares, A. (2017). Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants. En Plant Argonaute Proteins: Methods and Protocols. Springer Link. 93-112. https://doi.org/10.1007/978-1-4939-7165-7_693112Carbonell A, Carrington JC (2015) Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol 27:111–117. doi: 10.1016/j.pbi.2015.06.013Fang X, Qi Y (2016) RNAi in plants: an Argonaute-centered view. Plant Cell 28(2):272–285. doi: 10.1105/tpc.15.00920Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13(7):350–358. doi: 10.1016/j.tplants.2008.04.007Mallory A, Vaucheret H (2010) Form, function, and regulation of ARGONAUTE proteins. Plant Cell 22(12):3879–3889. doi: 10.1105/tpc.110.080671Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133(1):128–141. doi: 10.1016/j.cell.2008.02.033Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon GJ, Qi Y (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133(1):116–127. doi: 10.1016/j.cell.2008.02.034Zhang X, Niu D, Carbonell A, Wang A, Lee A, Tun V, Wang Z, Carrington JC, Chang CE, Jin H (2014) ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis. Nat Commun 5:5468. doi: 10.1038/ncomms6468Zhu H, Hu F, Wang R, Zhou X, Sze SH, Liou LW, Barefoot A, Dickman M, Zhang X (2011) Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145(2):242–256. doi: 10.1016/j.cell.2011.03.024Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y (2008) The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol 49(4):493–500. doi: 10.1093/pcp/pcn043Cuperus JT, Carbonell A, Fahlgren N, Garcia-Ruiz H, Burke RT, Takeda A, Sullivan CM, Gilbert SD, Montgomery TA, Carrington JC (2010) Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nat Struct Mol Biol 17(8):997–1003. doi: 10.1038/nsmb.1866Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y (2009) Rice MicroRNA effector complexes and targets. Plant Cell 21(11):3421–3435. doi: 10.1105/tpc.109.070938Carbonell A, Fahlgren N, Garcia-Ruiz H, Gilbert KB, Montgomery TA, Nguyen T, Cuperus JT, Carrington JC (2012) Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24(9):3613–3629. doi: 10.1105/tpc.112.099945Gilbert KB, Fahlgren N, Kasschau KD, Chapman EJ, Carrington JC, Carbonell A (2014) Preparation of multiplexed small RNA libraries from plants. Bio Protoc 4(21):e1275Wang L, Si Y, Dedow LK, Shao Y, Liu P, Brutnell TP (2011) A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq. PLoS One 6(10):e26426. doi: 10.1371/journal.pone.002642

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

Los viroides, los agentes infecciosos conocidos más simples, están constituidos por una molécula circular de RNA monocatenario. A pesar de su pequeño tamaño (246-401 nt) y de no codificar proteínas, son capaces de replicarse autónomamente, moverse sistémicamente, y causar enfermedades en ciertas plantas. En el presente trabajo hemos profundizado en el estudio de dos metodologías para el control de viroides basadas en ribozimas de cabeza de martillo con motivos de estabilización terciaria, y en RNAs interferentes que inducen en la planta una respuesta defensiva de silenciamiento génico mediado por RNA. Las ribozimas derivadas del viroide latente de la berenjena (Eggplant latent viroid, ELVd), que en su contexto natural median el autocorte de los RNAs multiméricos generados por un mecanismo de replicación de círculo rodante, son particularmente interesantes para adaptarlas a un formato trans. Hemos observado que la secuencia del trinucleótido que precede el sitio de autocorte de la ribozima de polaridad (+) del ELVd afecta a su actividad autocatalítica. Las constantes catalíticas de distintas variantes de este trinucleótido (AUA, AUC, GUA, GUC) determinadas in vitro a baja concentración de magnesio son diferentes en condiciones co- y postranscripcionales. Estos resultados sugieren que la ribozima de polaridad (+) del ELVd, y muy posiblemente otras ribozimas de cabeza de martillo naturales, han sido evolutivamente seleccionadas para actuar durante la transcripción de los RNAs viroidales, y que el trinucleótido AUC que precede al sitio de autocorte no se encuentra en la mayoría de las ribozimas de cabeza de martillo naturales por favorecer la adopción de estructuras metaestables catalíticamente inactivas durante la transcripción. La variante ELVd(+)-GUC, que presenta una constante catalítica alta e induce el corte de una amplia fracción del transcrito primario, fue seleccionada para diseñar variantes en trans frente al viroide del tubérculo fusiforme de la patata (PotatCarbonell Olivares, A. (2008). Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/2005Palanci

Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana

[EN] Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Roseal to obtain recombinant clone ZYMV-Rosl. While in some plant-virus combinations Roseal induces accumulation of anthocyanins in infected tissues, ZYMV-Rosl infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmen-tation in infected tissues of the model plant Nicotiana ben-thamiana. In this species, ZYMV-Rosl multiplied efficiently in local inoculated tissue but only a few progeny particles estab-lished infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Rosl local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Rosl systemic accumula-tion was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Rosl systemic accumulation dra-matically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.We thank V. Aragones for excellent technical assistance. This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO) through grants BI02014-54269-R and AGL2013-49919-EXP and by the Greek Ministry for Education and Religious Affairs (Program Aristeia II, 4499, ViroidmiR; ESPA 2007-2013). A. Carbonell was supported by an Individual Fellowship from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 655841.Cordero-Cucart, MT.; Cerdán García, L.; Carbonell Olivares, A.; Katsarou, K.; Kalantidis, K.; Daros Arnau, JA. (2016). Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 30(1):63-71. https://doi.org/10.1094/MPMI-11-16-0239-RS637130

Test-Retest reliability of Biodex Balance SD on physically active old people

The purpose of this study was to determine the reliability of the Biodex Balance System in elderly. Forty-five subjects aged 66±5.5 years old and weight 71.6±9.8 kg were tested on the Biodex Balance System. In order to calculate the reliability, the Fall Risk Test (FRt) and the Postural Stability Test (PSt) were measured on two separate occasions 7 days apart. Every subject completed the Falls Efficacy Scale-International (FES-I) questionnaire the first day of testing. The Fall Risk Index (FRi) showed a good ICC (.80) and a low percentage of variation of method error. The Overall Stability Index (OSi) showed a good and acceptable reliability measured by the ICC (.69) but a percentage of variation of method error near to 25%. FES-I Score was 23.1 (±7.2). The reliability of the BBS using Bland-Altman method showed that systematic errors (mean difference between test-retest) for the balance test developed were nearly zero and the 95% limits of agreement narrow, indicating a good reliability of the measurement. Biodex balance measures were showed reliable and may be useful for measuring the risk of falls and monitoring programs for prevent falls in elderly. This study revealed that fall risk assessment in older people must be incorporated into the evaluation process of the physical functioning.This study was in part supported by grants from the Spanish Ministry of Education (AP-2006-03676) and Science and Innovation (BES-2009-013442)

Asientos más seguros para trabajos en altura

The Instituto of Biomecánica, at the request of Comissions Obreres del País Valencià and under the funding of the Fundación para la Prevención de Riesgos Laborales, has carried out a study whose main objective has been to improve the culture of prevention among workers in rope access work.El Instituto de Biomecánica (IBV), a partir de la solicitud de Comissions Obreres del País Valencià (CC. OO.-CV) y con la financiación de la Fundación para la Prevención de Riesgos Laborales (FPRL), ha llevado a cabo un estudio cuyo objetivo ha consistido en mejorar la cultura de la prevención entre los trabajadores del sector de trabajos verticales

Safest seats in rope access work

[EN] The Instituto of Biomecánica, at the request of Comissions Obreres del País Valencià and under the funding of the Fundación para la Prevención de Riesgos Laborales, has carried out a study whose main objective has been to improve the culture of prevention among workers in rope access work.[ES] El Instituto de Biomecánica (IBV), a partir de la solicitud de Comissions Obreres del País Valencià (CC. OO.-CV) y con la financiación de la Fundación para la Prevención de Riesgos Laborales (FPRL), ha llevado a cabo un estudio cuyo objetivo ha consistido en mejorar la cultura de la prevención entre los trabajadores del sector de trabajos verticales.La actuación ha sido financiada por la Fundación para la Prevención de Riesgos Laborales a través de su convocatoria de asignación de recursos para acciones indirectas territoriales correspondientes al ejercicio 2010Palomares Olivares, N.; Cortés Carbonell, GJ.; Navarro Garcia, FJ.; Ferreras Remesal, A.; Oltra Pastor, A.; Ruiz Folgado, R.; Garcia Molina, C.... (2012). Asientos más seguros para trabajos en altura. Revista de biomecánica. (58):55-57. http://hdl.handle.net/10251/38478S55575

Libro de Proyectos Finales 2021 primer semestre

PregradoIngeniero CivilIngeniero de SistemasIngeniero ElectricistaIngeniero ElectrónicoIngeniero IndustrialIngeniero Mecánic