Hipusinación del factor de traducción eIF5A dependiente de poliaminas

RESUMEN La hipusinación es una modificación post-traduccional dependiente de espermidina que activa al factor de traducción eIF5A, y que es esencial en todos los eucariotas. En los últimos años se ha sugerido un importante papel para eIF5A en los procesos de senescencia y respuesta a estrés ambiental en plantas, en el establecimiento de la polaridad celular en levadura y su implicación en enfermedades tales como diabetes, VIH-1 o cáncer en humanos. Con el objetivo de caracterizar a nivel molecular la actividad biológica de eIF5A en plantas, hemos establecido una metodología basada en técnicas bioquímicas e inmunológicas para determinar el patrón de hipusinación de eIF5A en A. thaliana. La puesta a punto de esta metodología nos ha permitido demostrar que el tratamiento con ácido abscísico inhibe la activación por hipusinación de la isoforma eIF5A1. Además, para tratar de estudiar la función de eIF5A durante el desarrollo de A. thaliana, hemos realizado estudios funcionales basados en la caracterización de plantas transgénicas capaces de desactivar genéticamente la ruta dependiente de eIF5A mediante ARN de interferencia, condicionado a la aplicación de dexametasona. La desactivación condicional de la enzima de hipusinación desoxihipusina sintasa, produjo alteraciones durante el desarrollo y en respuesta a condiciones adversas de crecimiento, tales como floración temprana, inhibición del crecimiento de la raíz, alteraciones en los pelos radiculares, ramificación exacerbada del tallo, presencia de elementos traqueales completamente lignificados en hipocotilos, niveles reducidos de óxido nítrico e hipersensibilidad al ácido abscísico, sal y glucosa. Recientemente se ha demostrado que eIF5A es necesario para la traducción de proteínas con más de 3 prolinas sucesivas en su secuencia. El análisis de ontología realizado reveló un enriquecimiento de proteínas con poli-prolinas entre las implicadas en la organización del citoesqueleto de actina. La alteración de la actividad de eIF5A provocó defectos en la estructuración de los filamentos de actina en A. thaliana, S. cerevisiae y H. sapiens. El estudio de mutantes termosensibles de levadura demostró el requerimiento de eIF5A durante el proceso de reproducción sexual a través de la traducción de la formina Bni1. Los experimentos de regulación traduccional en células HeLa demostraron que el silenciamiento vía ARN de interferencia de eIF5A1 provocaba un defecto en la tasa de traducción de la formina FMNL1 y la proteína ezrina. Estos resultados confirman que la actividad esencial de eIF5A en el ribosoma parece conservada en organismos eucariotas, y afecta fundamentalmente a proteínas con poli-prolinas implicadas en la organización del citoesqueleto de actina.Belda Palazón, B. (2014). Hipusinación del factor de traducción eIF5A dependiente de poliaminas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48474TESI

Polyamines as Quality Control Metabolites Operating at the Post-Transcriptional Level

[EN] Plant polyamines (PAs) have been assigned a large number of physiological functions with unknown molecular mechanisms in many cases. Among the most abundant and studied polyamines, two of them, namely spermidine (Spd) and thermospermine (Tspm), share some molecular functions related to quality control pathways for tightly regulated mRNAs at the level of translation. In this review, we focus on the roles of Tspm and Spd to facilitate the translation of mRNAs containing upstream ORFs (uORFs), premature stop codons, and ribosome stalling sequences that may block translation, thus preventing their degradation by quality control mechanisms such as the nonsense-mediated decay pathway and possible interactions with other mRNA quality surveillance pathways.A.F. was funded by the Spanish Ministry of Science, Innovation and Universities, grant number BIO2015-70483-R, and B.B.-P. was funded by the Generalitat Valenciana grant, VALi+d GVA APOSTD/2017/039. 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HOS1 promotes plant tolerance to low-energy stress via the SnRK1 protein kinase

Funding Information: We thank Vera Nunes from the IGC Model Organism Unit/Plant Facility for excellent plant management and Filip Rolland for kindly providing the and lines. This work was supported by FCT ‐ Fundação para a Ciência e a Tecnologia, I.P., through GREEN‐IT ‐ Bioresources for Sustainability R&D Unit (UIDB/04551/2020, UIDP/04551/2020), by the LS4FUTURE Associated Laboratory (LA/P/0087/2020, PTDC/BIA‐FBT/4942/2020, LISBOA‐01‐0145‐FEDER‐028128, PTDC/BIA‐BID/32347/2017, EXPL/ASP‐AGR/1329/2021, 2022.08339.PTDC, SFRH/BPD/116116/2016 [LM], PD/BD/114361/2016 [BP], and 2020.03177.CEECIND [EBG]), and by the European Union Horizon 2020 research and innovation program (Grant Agreement number: 867426 — ABA GrowthBalance — H2020‐WF‐2018‐2020/H2020‐WF‐01‐2018, awarded to BBP). NLS‐SnRK1α1 βMYR‐SnRK1α1 Publisher Copyright: © 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.Plants need to integrate internal and environmental signals to mount adequate stress responses. The NUCLEAR PORE COMPLEX (NPC) component HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) is emerging as such an integrator, affecting responses to cold, heat, light, and salinity. Stress conditions often converge in a low-energy signal that activates SUCROSE NON-FERMENTING 1-RELATED KINASE 1 (SnRK1) to promote stress tolerance and survival. Here, we explored the role of HOS1 in the SnRK1-dependent response to low-energy stress in Arabidopsis thaliana, using darkness as a treatment and a combination of genetic, biochemical, and phenotypic assays. We show that the induction of starvation genes and plant tolerance to prolonged darkness are defective in the hos1 mutant. HOS1 interacts physically with the SnRK1α1 catalytic subunit in yeast two-hybrid assays and in planta, and the nuclear accumulation of SnRK1α1 is reduced in the hos1 mutant. Likewise, another NPC mutant, nup160, exhibits lower activation of starvation genes and decreased tolerance to prolonged darkness. Importantly, defects in low-energy responses in the hos1 background are rescued by fusing SnRK1α1 to a potent nuclear localization signal or by sugar supplementation during the dark treatment. Altogether, this work demonstrates the importance of HOS1 for the nuclear accumulation of SnRK1α1, which is key for plant tolerance to low-energy conditions.publishersversionpublishe

Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus

Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA

[EN] Abscisic acid (ABA) is an essential hormone for plant development and stress responses. ABA signaling is suppressed by clade A PP2C phosphatases, which function as key repressors of this pathway through inhibiting ABA-activated SnRK2s (SNF1-related protein kinases). Upon ABA perception, the PYR/PYL/RCAR ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity. While thismechanismhas been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. Arabidopsis thaliana RING DOMAIN LIGASE5 (RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain unknown. We report that RGLG5, together with RGLG1, releases the PP2C blockade of ABA signaling by mediating PP2CA protein degradation. ABA promotes the interaction of PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for ABA-dependent PP2CA turnover. Downregulation of RGLG1 and RGLG5 stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the rglg1 amiR (artificial microRNA)-rglg5 pp2ca-1 triple mutant, supporting a functional link among these loci. Overall, our data indicate that RGLG1 and RGLG5 are important modulators of ABA signaling, and they unveil amechanismfor activation of the ABA pathway by controlling PP2C half-life.We thank Andreas Bachmair for the rglg1 mutant, Sean R. Cutler for the pyr1 pyl1 pyl2 pyl4 seeds, Dapeng Zhang for the transgenic material harboring ABI2, Hongwei Guo and Jianmin Zhou for the pCAMBIA1300-Nluc and pCAMBIA1300-Cluc vectors, and John Olson for assistance in English editing. Work in C.A.'s laboratory was supported by grants from the National Key Basic Science "973" Program (Grant 2012CB114006), the National Natural Science Foundation (Grants 31272023, 31170231, and 90817001) of the Chinese government, and by the State Key Laboratory of Protein and Plant Gene Research, Peking University. Work in P.L.R.'s laboratory was supported by Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Cientificas (Grant BIO2014-52537-R).Wu, Q.; Zhang, X.; Peirats-Llobet, M.; Belda Palazón, B.; Wang, X.; Cui, S.; Yu, X.... (2016). Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA. Plant Cell. 28(9):2178-2196. https://doi.org/10.1105/tpc.16.003642178219628

Citrus exocortis viroid causes ribosomal stress in tomato plants

[EN] Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.Spanish Ministry of Science, Innovation and Universities [BIO2009-11818, BIO2015-70483-R to A.F.]; Spanish Ministry of Science, Innovation and Universities [BFU2009-11958]; Generalitat Valenciana (Valencia, Spain) [AICO/2017/048]; Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Universite de Sherbrooke; J.-P.P. holds the Research Chair of the Universite de Sherbrooke in RNA Structure and Genomics, and is a member of the Centre de Recherche du CHUS; B.B.-P. was a recipient of a VALi+d postdoctoral contract of the Generalitat Valenciana [APOSTD/2017/039]; Schleiff group is funded through the Deutsche Forschungsgemeinschaft [SFB 902]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities.Cottilli, P.; Belda-Palazón, B.; Adkar-Purushothama, CR.; Perreault, J.; Schleiff, E.; Rodrigo Bravo, I.; Ferrando Monleón, AR.... (2019). Citrus exocortis viroid causes ribosomal stress in tomato plants. Nucleic Acids Research. 47(16):8649-8661. https://doi.org/10.1093/nar/gkz679S86498661471

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

[EN] Hormone- and stress-induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone-induced ubiquitination plays a crucial role to determine half-life of key negative regulators of hormone signaling. For ABA signaling, degradation of clade A PP2Cs, such as PP2CA or ABI1, is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of PP2C activity. ABA promotes the degradation of PP2CA through the RGLG1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG1 with PP2CA given they are predominantly found in plasma membrane and nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG1 and promotes nuclear interaction with PP2CA. We found RGLG1 is myristoylated in vivo, which facilitates its attachment to plasma membrane. ABA inhibits myristoylation of RGLG1 through downregulation of Nmyristoyltransferase1 (NMT1) and promotes nuclear translocation of RGLG1 in a cycloheximide-insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-receptor-phosphatase complexes. We show that RGLG1Gly2Ala -mutated in the Nterminal myristoylation site- shows constitutive nuclear localization and causes enhanced response to ABA and salt/osmotic stress. RGLG1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG1-PP2CA-PYL8. In summary, we provide evidence that an E3 ligase can dynamically re-localize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation.Work in P.L.R.'s laboratory was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas through grants BIO2014-52537-R and BIO2017-82503-R. This work was also funded by grants from the Deutsche Forschungsgemeinschaft (DFG) Ku931/4-1 to J. K., and BA4742/1-2 to O.B. B.B. was funded by Programa VALi+ d GVA APOSTD/2017/039. J.J. was supported by an FPI contract from MINECOBelda Palazón, B.; Julian, J.; Coego, A.; Wu, Q.; Zhang, X.; Batistic, O.; Alquraishi, SA.... (2019). ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA. 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Fertility and Polarized Cell Growth Depends on eIF5A for Translation of Polyproline-Rich Formins in Saccharomyces cerevisiae

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes.We thank M. E. Perez-Martinez and I. Quilis for their help in viability and beta-galactosidase experiments; J. E. Perez-Ortin for critical reading of the manuscript; and F. Randez-Gil, G. Ammerer, J. Warringer, and S. R. Valentini for materials. We acknowledge funding from the Spanish MCINN (BFU2010-21975-C03-01), the Generalitat Valenciana (PROMETEO 2011/088 and ACOMP/2012/001), Universitat de Valencia (UV-INV-AE13-139034), and support from European Union funds (FEDER). T. L. and B. B.- P. are recipients of a PROMETEO and a VALi+d predoctoral (ACIF2010/085) contract, respectively, from the Generalitat Valenciana.Li, T.; Belda Palazón, B.; Ferrando Monleón, AR.; Alepuz, P. (2014). Fertility and Polarized Cell Growth Depends on eIF5A for Translation of Polyproline-Rich Formins in Saccharomyces cerevisiae. Genetics. 197(4):1191-1200. doi:10.1534/genetics.114.166926S11911200197

ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses such as drought, partly by modulating growth through poorly understood mechanisms. Here, we show that ABA-triggered repression of cell proliferation in the Arabidopsis thaliana root meristem relies on the swift subcellular relocalization of SNF1-RELATED KINASE 1 (SnRK1). Under favorable conditions, the SnRK1 catalytic subunit, SnRK1 alpha 1, is enriched in the nuclei of root cells, and this is accompanied by normal cell proliferation and meristem size. Depletion of two key drivers of ABA signaling, SnRK2.2 and SnRK2.3, causes constitutive cytoplasmic localization of SnRK1 alpha 1 and reduced meristem size, suggesting that, under nonstress conditions, SnRK2s promote growth by retaining SnRK1 alpha 1 in the nucleus. In response to ABA, SnRK1 alpha 1 translocates to the cytoplasm, and this is accompanied by inhibition of target of rapamycin (TOR), decreased cell proliferation, and reduced meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK1 alpha 1 relocalization to the cytoplasm and ABA-elicited inhibition of TOR. Furthermore, fusing SnRK1 alpha 1 to an SV40 nuclear localization signal leads to defective ABA-dependent TOR repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK1 alpha 1 subcellular localization are crucial for inhibiting TOR and root growth in response to ABA. Rapid relocalization of central regulators such as SnRK1 may represent a general strategy of eukaryotic organisms to respond to environmental changes

Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus