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In recent years, miR528, a monocot-specific miRNA, has been assigned multifaceted roles during development and stress response in several plant species. However, the transcription regulation and the molecular mechanisms controlling

Trypanosoma cruzi is the etiological agent of Chagas disease, whose clinical outcome ranges from asymptomatic individuals to chronic fatal megasyndromes. Despite being central to pathogenesis, the regulation of parasite virulence factors’ expression remains largely unknown. In this work, the relative expression of several parasite virulence factors between two TcI strains (Ninoa, low virulence and Qro, high virulence) was assessed by qRT-PCR of total and of polysome-associated mRNA, as well as by western blots. Trypomastigotes were also incubated with specific anti-sense morpholino oligonucleotides to block the translation of a selected virulence factor, calreticulin, in both strains. Ninoa trypomastigotes showed significantly lower levels of trypomastigote-decay acceleration factor, complement regulatory protein, complement C2 receptor inhibitor trispanning, and glycoproteins 82 and 90 mRNAs compared with Qro. There was a significantly lower recruitment of complement regulatory protein and complement C2 receptor inhibitor trispanning mRNAs to polysomes and higher recruitment of MASP mRNA to monosomes in Ninoa strain. Calreticulin mRNA displayed both a higher total mRNA level and recruitment to translationally active polysomes in the Ninoa strain (low virulence) than in the Qro strain (high virulence). When calreticulin was downregulated by ≈ 50% by anti-sense morpholino oligonucleotides, a significant decrease of parasite invasion in mammalian cells was found in both strains. Calreticulin downregulation, however, only increased significantly the activation of the complement system by Ninoa trypomastigotes. These results suggest a role for the regulation of virulence factors’ gene expression in the differential virulence among T. cruzi strains. Furthermore, a possible function of calreticulin in parasite invasion not related to its binding to complement factors is shown.

Aim: We aimed to determine the functionality of rotavirus NSP3 dimers. Materials & methods: We expressed rhesus rotavirus NSP3 and determined the kinetics of host cell translation inhibition and the levels of accumulated dimerization intermediates and dimers. Results: We observed a linear kinetics of host cell translation inhibition, which correlated well with the sum of the dimerization intermediates and dimers. Treatment with 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced the accumulation of NSP3 dimers and potentiated host cell translation inhibition. Conclusion: Our results show that NSP3 dimer formation does not correlate with host cell translation inhibition and suggest that both NSP3 dimers and dimerization intermediates are functional and inhibit host cell translation.

Summary Spermidine (Spd) is a nitrogen sink and signaling molecule that plays pivotal roles in eukaryotic cell growth and must be finetuned to meet various energy demands. In eukaryotes, target of rapamycin (TOR) is a central nutrient sensor, especially N, and a master-regulator of growth and development. Here, we discovered that Spd stimulates the growth of maize and Arabidopsis seedlings through TOR signaling. Inhibition of Spd biosynthesis led to TOR inactivation and growth defects. Furthermore, disruption of a TOR complex partner RAPTOR1B abolished seedling growth stimulation by Spd. Strikingly, TOR activated by Spd promotes translation of key metabolic enzyme upstream open reading frame (uORF)-containing mRNAs, PAO and CuAO, by facilitating translation reinitiation and providing feedback to polyamine metabolism and TOR activation. The Spd-TOR relay protected young-age seedlings of maize from expeditious stress heat shock. Our results demonstrate Spd is an upstream effector of TOR kinase in planta and provide its potential application for crop protection., Graphical abstract, Highlights • Spermidine (Spd) stimulates growth of maize and Arabidopsis by activating TOR signaling • TOR stimulates translation efficiency of uORF-containing mRNAs involved in Spd catabolism • TOR provides feedback to polyamine homeostasis in response to excess of Spd • The Spd-TOR signaling axis protects maize seedlings from expeditious heat stress, Cell biology; Plant biology; Plant physiology

Plant defense and adaptation to adverse environmental conditions rely on gene expression control, such as mRNA transcription, processing, stability, and translation. Sudden temperature changes are common in the era of global warming; thus, understanding plant acclimation responses at the molecular level becomes imperative. mRNA translation initiation regulation has a pivotal role in achieving the synthesis of the appropriate battery of proteins needed to cope with temperature stress. In this study, we analyzed the role of translation initiation factors belonging to the eIF4E family in Arabidopsis acclimation to cold temperatures and freezing tolerance. Using knockout (KO) and overexpressing mutants of AteIF4E1 or AteIF(iso)4E, we found that AteIF4E1 but not AteIF(iso)4E overexpressing lines displayed enhanced tolerance to freezing without previous acclimation at 4°C. However, KO mutant lines, eif(iso)4e-1 and eif4e1-KO, were more sensitive to the stress. Cold acclimation in wild-type plants was accompanied by increased levels of eIF4E1 and eIF(iso)4E transcript levels, polysomes (P) enrichment, and shifts of these factors from translationally non-active to active fractions. Transcripts, previously found as candidates for eIF(iso)4E or eIF4E1 selective translation, changed their distribution in both P and total RNA in the presence of cold. Some of these transcripts changed their polysomal distribution in the mutant and one eIF4E1 overexpressing line. According to this, we propose a role of eIF4E1 and eIF(iso)4E in cold acclimation and freezing tolerance by regulating the expression of stress-related genes.

Maize somatic embryogenesis (SE) requires the induction of embryogenic callus and establishment of proliferation before plant regeneration. The molecular mechanisms underlying callus embryogenic potential are not well understood. Here we explored the role of small RNAs (sRNAs) and the accumulation of their target transcripts in maize SE at the dedifferentiation step using VS-535 zygotic embryos collected at distinct developmental stages and displaying contrasting in vitro embryogenic potential and morphology. MicroRNAs (miRNAs), trans-acting siRNAs (tasiRNAs), heterochromatic siRNAs (hc-siRNAs) populations and their RNA targets were analyzed by high-throughput sequencing. Abundances of specific miRNAs, tasiRNAs and targets were validated by qRT-PCR. Unique accumulation patterns were found for immature embryo at 15 Days After Pollination (DAP) and for the callus induction from this explant, as compared to 23 DAP and mature embryos. miR156, miR164, miR166, tasiARFs and the 24 nt hc-siRNAs displayed the most strikingly different patterns between explants and during dedifferentiation. According to their role in auxin responses and developmental cues, we conclude that sRNA-target regulation operating within the 15 DAP immature embryo explant provides key molecular hints as to why this stage is relevant for callus induction with successful proliferation and plant regeneration.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets, however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Plants make decisions throughout their lifetime based on complex networks. Phase transitions during seed growth are not an exception. From embryo development through seedling growth, several molecular pathways control genome stability, environmental signal transduction and the transcriptional landscape. Particularly, epigenetic modifications and small non-coding RNAs (sRNAs) have been extensively studied as significant handlers of these processes in plants. Here, we review key epigenetic (histone modifications and methylation patterns) and sRNA-mediated regulatory networks involved in the progression from seed maturation to germination, their relationship with seed traits and crosstalk with environmental inputs.

The Glucose-Target of Rapamycin (Glc-TOR) pathway has been studied in different biological systems, but scarcely during early seed germination. This work examines its importance for cell proliferation, expression of cell cycle key genes, their protein levels, besides morphology and cellularization of the root apical meristem of maize (Zea mays) embryo axes during germination under the influence of two simple sugars, glucose and sucrose, and a specific inhibitor of TOR activity, AZD 8055. The two sugars promote germination similarly and to an extent, independently of TOR activity. However, the Glc-TOR pathway increases the number of cells committed to proliferation, increasing the expression of a cell cycle gene, ZmCycD4;2, a putative G1/S regulator. Also, Glc-TOR may have influence on the protein stability of another G1/S cyclin, ZmCycD3, but had no influence on ZmCDKA;1 or ZmKRP3 or their proteins. Results suggest that the Glc-TOR pathway participates in the regulation of proliferation through different mechanisms that, in the end, modify the timing of seed germination.

Agaves resist extreme heat and drought. In A. tequilana var. azul, the central spike of the rosette -containing the shoot apical meristem and folded leaves in early stages of development- is remarkably heat tolerant. We found that the most abundant protein in this organ is a 27 kDa protein. This protein was named mayahuelin to honor Mayahuel, the agave goddess in the Aztec pantheon. LC-MS/MS analyses identified mayahuelin as a type I RIP (Ribosome Inactivating Protein). In addition to the spike, mayahuelin was expressed in the peduncle and in seeds, whereas in mature leaves, anthers, filaments, pistils, and tepals was absent. Anti-mayahuelin antibody raised against the A. tequilana var. azul protein revealed strong signals in spike leaves of A. angustifolia, A. bracteosa, A. rhodacantha, and A. vilmoriniana, and moderate signals in A. isthmensis, A. kerchovei, A. striata ssp. falcata, and A. titanota, indicating conservation at the protein level throughout the Agave genus. As in charybdin, a type I RIP characterized in Drimia maritima, mayahuelin from A. tequilana var. azul contains a natural aa substitution (Y76D) in one out of four aa comprising the active site. The RIP gene family in A. tequilana var. azul consists of at least 12 genes and Mayahuelin is the only member encoding active site substitutions. Unlike canonical plant RIPs, expression of Mayahuelin gene in S. cerevisiae did not compromise growth. The inhibitory activity of the purified protein on a wheat germ in vitro translation system was moderate. Mayahuelin orthologs from other Agave species displayed one of six alleles at Y76: (Y/Y, D/D, S/S, Y/D, Y/S, D/S) and proved to be useful markers for phylogenetic analysis. Homozygous alleles were more frequent in wild accessions whereas heterozygous alleles were more frequent in cultivars. Mayahuelin sequences from different wild populations of A. angustifolia and A. rhodacantha allowed the identification of accessions closely related to azul, manso, siguin, mano larga, and bermejo varieties of A. tequilana and var. espadin of A. angustifolia. Four A. rhodacantha accessions and A. angustifolia var. espadin were closer relatives of A. tequilana var. azul than A. angustifolia wild accessions or other A. tequilana varieties.

Alternatives to the cap mechanism in translation are often used by viruses and cells to allow them to synthesize proteins in events of stress and viral infection. In Drosophila there are hundreds of polycistronic messenger RNA (mRNA), and various mechanisms are known to achieve this. However, proteins in a same mRNA often work in the same cellular mechanism, this is not the case for Drosophila's Swc6/p18Hamlet homolog Dmp18, part of the SWR1 chromatin remodeling complex, who is encoded in a bicistronic mRNA next to Dmp8 (Dmp8-Dmp18 transcript), a structural component of transcription factor TFIIH. The organization of these two genes as a bicistron is conserved in all arthropods, however the length of the intercistronic sequence varies from more than 90 to 2 bases, suggesting an unusual translation mechanism for the second open reading frame. We found that even though translation of Dmp18 occurs independently from that of Dmp8, it is necessary for Dmp18 to be in that conformation to allow its correct translation during cellular stress caused by damage via heat-shock and UV radiation.

In vitro plant regeneration addresses basic questions of molecular reprogramming in the absence of embryonic positional cues. The process is highly dependent on the genotype and explant characteristics. However, the regulatory mechanisms operating during organ differentiation from in vitro cultures remain largely unknown. Recently, miRNAs have emerged as key regulators during embryogenic callus induction, plant differentiation, auxin responses and totipotency. Here, we explored how development-related miRNA switches the impact on their target regulation depending on physiological and molecular events taking place during maize Tuxpe&ntilde, o VS-535 in vitro plant regeneration. Three callus types with distinctive regeneration potential were characterized by microscopy and histological preparations. The embryogenic calli (EC) showed higher miRNA levels than non-embryogenic tissues (NEC). An inverse correlation for miR160 and miR166 targets was found during EC callus induction, whereas miR156, miR164 and miR394 displayed similar to their targets RNA accumulation levels. Most miRNA accumulation switches took place early at regenerative spots coincident with shoot apical meristem (SAM) establishment, whereas miR156, miR160 and miR166 increased at further differentiation stages. Our data uncover particular miRNA-mediated regulation operating for maize embryogenic tissues, supporting their regulatory role in early SAM establishment and basipetala growth during the in vitro regeneration process.

To assure an efficient protein production under stress conditions, some transcripts are modulated at the translational level. To better understand the control of protein production under stress, we analysed the enhancing effect of the 3′ untranslated region (UTR) of the water deficit responsive gene PvLEA6 from Phaseolus vulgaris on the regulation of its expression. GUS activity and transcript loading on polysomes were analysed using Arabidopsis transgenic plants with constructs containing GUS open reading frame fused to wild type or mutated PvLEA6-3′UTR, driven by the PvLEA6 promoter, grown under optimal or stress conditions. The effect of the PvLEA6-3′UTR on gene transcription and transcript stability was discarded. We demonstrated that the PvLEA6-3′UTR allows preferential polysome loading of the GUS reporter transcript under water deficit. The effect of this region on protein synthesis was also supported by in vitro translation data. Particular conserved motifs in this region responsible for translation stimulation were identified. Specific interaction of the 3′UTR with cellular protein(s) was shown. Our data support a specific role of PvLEA6-3′UTR leading to efficient protein synthesis and hence a competent response under water deficit and suggest the participation of mRNA binding proteins in translational enhancement of the PvLEA6 mRNA.

In plants, differentiated somatic cells can revert their identity to pluripotent, reprogrammed cells in order to optimize growth and development depending on external conditions and in aid of overcoming their limitations as sessile organisms. Different modes of regeneration include tissue repair, de novo organogenesis and somatic embryogenesis (SE). The latter usually comprise the formation of proliferating pluripotent cell masses called callus. Identification and characterization of genes involved in the SE process allows the exploitation of distinctive features that make a tissue susceptible to change its normal cell fate and produce new plants massively.

MicroRNAs are tiny molecules that strikingly change their expression patterns and distribution during somatic embryogenesis induction and plant regeneration. It is of great relevance to analyze simultaneously the microRNA and target mRNA fates to understand their role in promoting an adequate embryogenic response to external stimulus in the regenerating tissues. Here we describe a method to evaluate the expression patterns of miRNAs or other sRNAs and their target regulation in distinctive tissues observed during maize plant regeneration. Key features of the method include the classification of regenerating plant material with reproducibly distinctive morphological characteristics and a purification procedure that renders high-quality small and large RNA separation from the same sample for qRT-PCR analysis.

Marine sandy beaches represent dynamic environments often subject to harsh conditions and climate fluctuations, where natural and anthropogenic inputs of freshwater from fluvial and pluvial sources alter salinity, which has been recognized as a key variable affecting the distribution of aquatic organisms and influencing critical physiological processes. The marine arenicolous fungus Corollospora maritima is a worldwide-distributed saprobe that has been reported to present tolerance to freshwater. Here, we present a transcriptome analysis that will provide the first insight of the genomic content for this fungus and a gene expression comparison between two different salinity conditions. We also identified genes that are candidates for being differentially expressed in response to environmental variations on salinity during the fungal growth. The de novo reconstruction of C. maritima transcriptome Illumina sequencing provided a total of 14,530 transcripts (16 megabases). The comparison between the two growth conditions rendered 103 genes specifically overexpressed in seawater, and 132 genes specifically up-regulated under freshwater. Using fungal isolates collected from different beaches, the specific environmental regulation of particular transcript differential expression was confirmed by RT-qPCR. To our knowledge, this is the first analysis that explores the marine fungus C. maritima molecular responses to overcome freshwater stress, and these data could shed light to understand the fungal adaptation and plasticity mechanisms to the marine habitat.

Barley malting quality depends on seed characteristics achieved during grain development and germination. One important parameter is protein accumulation in the mature seed, which may vary between cultivars. Here we conducted a protein pattern analysis in the range of pI 4–7 of mature grains from five Mexican barley cultivars, commonly used for malt and beer production. Reproducibly distinct protein spots, separated by 2D SDS PAGE, were identified by mass spectrometry and considered as potential markers for cultivars with distinct seed protein accumulation. The expression patterns of glutamate decarboxylase (GAD) and protein disulfide isomerase (PDI1-1) were followed at transcript level during grain development for three independent growth cycles to establish whether differences between cultivars were reproducible. Quantitative determination of PDI1-1 protein levels by ELISA confirmed a reproducibly, distinctive accumulation and post-translational modifications between cultivars, which were independent of plant growth regimes. According to its impact on differential storage protein accumulation, we propose the PDI1-1 protein as potential biomarker for Mexican malting barley cultivars.

Somatic embryogenesis represents an alternative developmental process used to achieve genetic transformation and to approach key questions in maize development. It is known that embryogenic callus induction and plant regeneration are accompanied by microRNA expression changes. However, small RNA (sRNA) populations have not been explored during the proliferative callus subculture establishment and their impact on maintaining the dedifferentiated status and embryogenic potential is far from being completely understood. Here we globally tested the sRNA populations in explants (immature embryos), induced and established maize embryogenic callus from the Mexican cultivar VS-535, Tuxpeno landrace. We detected readjustments in 24 nt and 21–22 nt sRNAs during the embryogenic callus (EC) establishment and maintenance. A follow up on specific microRNAs (miRNAs) indicated that miRNAs related to stress response substantially increase upon the callus proliferation establishment, correlating with a reduction in some of their target levels. On the other hand, while 24 nt-long heterochromatic small interfering RNAs (hc-siRNAs) derived from transposable retroelements transiently decreased in abundance during the EC establishment, a population of 22 nt-hc-siRNAs increased. This was accompanied by reduction in transposon expression in the established callus subcultures. We conclude that stress- and development-related miRNAs are highly expressed upon maize EC callus induction and during maintenance of the subcultures, while miRNAs involved in hormone response only transiently increase during induction. In addition, the establishment of a proliferative status in embryogenic callus is accompanied by important readjustments in hc-siRNAs mapping to long tandem repeat (LTR) retrotransposons, and their expression regulation.

Apoptosis is an important mechanism for maintaining germ line health. In Caenorhabditis elegans, germ cell apoptosis occurs under normal conditions to sustain gonad homeostasis and oocyte quality. Under stress, germ cell apoptosis can be triggered via different pathways, including the following: (i) the CEP-1/p53 pathway, which induces germ cell apoptosis when animals are exposed to DNA damage; (ii) the mitogen-activated protein kinase kinase (MAPKK) pathway, which triggers germ cell apoptosis when animals are exposed to heat shock, oxidative stress, or osmotic stress; and (iii) an unknown mechanism that triggers germ cell apoptosis during starvation. Here, we address how starvation induces germ cell apoptosis. Using polysomal profiling, we found that starvation for 6 h reduces the translationally active ribosomes, which differentially affect the mRNAs of the core apoptotic machinery and some of its regulators. During starvation, lin-35/Rb mRNA increases its expression, resulting in the accumulation of this protein. As a consequence, LIN-35 downregulates the expression of the antiapoptotic gene ced-9/Bcl-2. We observed that the reduced translation of ced-9/Bcl-2 mRNA during food deprivation together with its downregulation drastically affects its protein accumulation. We propose that CED-9/Bcl-2 downregulation via LIN-35/Rb triggers germ cell apoptosis in C. elegans in response to starvation.

During seed germination, metabolism is reactivated, DNA is repaired and cell division is restarted in the meristems. The mechanisms that co-ordinate cell growth and division in maize embryonic axes during germination are not well understood. However, the presence of a factor similar to IGF (insulin-like growth factor) that accelerates germination has been reported. In the present work, the regulation of the cell-cycle restart by bovine insulin [which has been demonstrated to produce similar effects as insulin-like growth factor of maize (ZmIGF) in maize seeds] was studied in germinating embryonic axes. Our results showed that bovine insulin differentially stimulates growth, S6K phosphorylation,S6rptranscript accumulation on the polysomal fraction, as well asde novoDNA synthesis in the radicles and the coleoptiles of the embryonic axis. A stronger and earlier effect was observed in radicles compared to coleoptiles; therefore, the effect of insulin on the cell cycle of the root meristem was studied by flow cytometry. The G1–S transition was stimulated and cell proliferation was induced. Furthermore, it was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) that bovine insulin increased E2F and PCNA (proliferating cell nuclear antigen) transcription after 15 h of germination and PCNAde novosynthesis at 15 h of germination. These results show that bovine insulin preferentially stimulates growth in the radicles of germinating embryonic axes and suggest that its effect on the G1–S transition and the activation of cell proliferation is mediated by the induction of E2F and PCNA transcription.

A characteristic mechanism of gene expression regulation during seed germination is the selective translation of mRNAs. Previous findings indicate that the two cap-binding complexes eIF4F (with eIF4E and eIF4G subunits) and eIF(iso)4F [with eIF(iso)4E and eIF(iso)4G subunits] are differentially expressed during maize seed germination. In addition, several studiesin vitrohave suggested that these factors may participate in selective mRNA translation. The translational activities of eIF4E and eIF(iso)4E were testedin vitrousing transcripts from two different sets: dry (0 h) and 24-h-imbibed maize embryonic axes.In vitrotranslation of these mRNA pools in the presence of the recombinant eIF4E or eIF(iso)4E, and the native cap-binding complexes from dry- or 24-h-imbibed axes, produced different profiles of proteins which were visualized by two-dimensional protein gels and autoradiography. The data indicated that eIF(iso)4E was particularly required for translation of the stored mRNAs from dry seeds, and that eIF4E was unable to fully replace the eIF(iso)4E activity. In addition, the dry seed mRNA pool was translated by the cap-binding complex isolated from dry seeds better than by the complex isolated from 24-h-imbibed seeds, whereas the translational efficiency of the mRNA pool from 24-h-imbibed seeds was similar between the cap-binding complexes from these two stages. Interestingly, eIF(iso)4E was more abundant than eIF4E in dry seeds, while both cap-binding proteins were present at similar levels in 24-h-imbibed seeds. These results suggest that the ratio of eIF(iso)4E to eIF4E in the corresponding eIF4F complex is critical for the mechanisms of translational control during maize germination.

El norteamericano Roger D. Kornberg recibió el pasado diciembre el Premio Nobel de Química por sus trabajos sobre uno de los elementos clave de la vida, la transcripción de los genes, siguiendo así las huellas de su padre, Arthur Kornberg, Premio Nobel de Medicina hace cerca de medio siglo. Sus investigaciones le han permitido ser el primero en explicar «la historia familiar sobre la vida», según definición de la Real Academia Sueca de las Ciencias. Kornberg decodificó el proceso de copiado de la información genética contenida en la molécula de ADN a una molécula intermediaria llamada ARN mensajero en el grupo de organismos denominados eucariontes (cuyas células tienen núcleo delimitado y de los cuales los humanos formamos parte). Para que el cuerpo pueda utilizar la información almacenada en sus genes es necesario primero hacer una copia de esa información en el núcleo y transferirla hacia el exterior, donde es utilizada para la producción de proteínas.

Recruitment of eukaryotic mRNAs by the translation machinery requires binding of their 5′ end (cap) to eIF4E in complex with the bridge protein eIF4G (eIF4F). Plant eIF(iso)4E and eIF(iso)4G form the eIF(iso)4F complex, distinct from eIF4F (formed by eIF4E and eIF4G). Separate eIF(iso)4E and eIF4E clades are detected upon seed plants’ arrival in evolution, while eIF(iso)4G has been present since green algae. Strong preference is observed between eIF(iso)4G and eIF(iso)4E, as well as between eIF4E and eIF4G in vitro and in vivo, suggesting that these complexes exert functionally distinct roles in seed plants. However, the regulatory mechanisms underlying their role in translation or other cellular processes remain mainly unknown, with the notable exception of plant-virus interaction. Individual isoforms are required in a specific way for successful virus infection in a range of plant species. The isolation of resistance-associated mutations in eIF4E family members pinpointed their role through direct interaction with the viral genome-linked protein VPg. In this chapter we review recent research on eIF4E diversification with emphasis on their cellular function and roles in the specificity of plant-virus interaction.

Insulin and ‘insulin-like’ growth factors (IGFs) are known to regulate cell growth in eukaryotes by stimulating a signal transduction pathway that exerts translational control. Intermediate kinases of this pathway, target of rapamycin (TOR) and ribosomal protein S6 kinase (S6K), have been reported in Arabidospsis thaliana and Zea mays. However, upstream signal inducers and downstream targets of the pathway are not well known in plants. The objective of this work is to inquire whether plant growth is regulated by a signal transduction pathway similar to the insulin/IGF-stimulated pathway in other metazoans. Insulin as well as Zea mays insulin-related peptide (ZmIGF), which is a maize, 20-kDa peptide fraction recognized by insulin antibody, were used as effectors to stimulate maize axes growth from germinating seeds. ZmIGF expression was identified in axes from germinating maize seeds and immunolocalized in the meristems of these tissues. Significant enhancement of specific de novo protein synthesis of the translational apparatus components was found in the stimulated axes. Reverse-transcription-polymerase chain reaction analysis of total and polysomal RNA pools in ZmIGF- or insulin-stimulated axes confirmed these data by revealing specific mRNA recruitment into polysomes. In addition, the same stimuli induced activation of S6 ribosomal protein kinase (ZmS6K) in germinating maize axes. All the above effects were inhibited by rapamycin, indicating that they depend on TOR activity. We conclude that a TOR–S6K signal transduction pathway is functional in maize germination, as that found for non-photosynthetic eukaryotes. The evolutionary implications of these findings are discussed.

*Summary Maize embryonic axes contain stored mRNAs, some of which are able to undergo cap-independent translation initiation during germination. The Hsp101 mRNA, encoding a heat shock protein, is essential for thermotolerance induction and is present among the stored transcripts. This research aimed to investigate whether the Hsp101 transcript is IRES-driven regulated upon heat stress. Hsp101 transcribed either in vitro or in vivo was efficiently translated via a cap-independent mechanism. This was observed either in an animal in vitro translation system containing proteolytically cleaved eukaryotic initiation factor eIF4G or in a plant system lacking both eIF4E and eIFiso4E initiation factors. Deletion of the 5¢ untranslated region (UTR) from the Hsp101 mRNA abolished its cap-independent translation indicating that this nucleotide sequence is required to confer cap-independent initiation. Bicistronic constructs containing the Hsp101 mRNA 5¢UTR in sense and anti-sense directions between two reporter genes were translated in both cap-independent systems. A similar bicistronic construct containing a viral internal ribosome entry site (IRES) element between the reporter genes was used as control. Internal translation of the second reporter gene was observed when the Hsp101 5¢UTR was in the sense but not in the anti-sense orientation in the bicistronic construct. Taken together, these data suggest that the 5¢UTR of maize Hsp101, a plant cellular mRNA, functions as an IRES-like element accounting for its capindependent translation during heat stress.

The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6-20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.

Auxin is known to stimulate protein synthesis in many plant tissues, but the mechanisms involved in this process are unknown. The present research inquires whether auxin might regulate selective translation of mRNAs by inducing S6 ribosomal protein phosphorylation on the 40S ribosomal subunit in maize (Zea mays L.). Maize embryonic axes auxin-stimulated by natural (IAA) or synthetic (Dicamba or 1-NAA) auxins, selectively increased ribosomal protein synthesis. This effect was not reproduced by auxin inactive analogue 2-NAA. Enhanced S6 ribosomal protein phosphorylation on the 40S ribosomal subunit was also observed after auxin stimulation, as measured by [32P] incorporation into this protein. This increment did not occur when stimulation was performed with the inactive auxin analogue. Further, increased recruitment into polysomes of two 5'TOP-like mRNAs, encoding for the initiation translation factor eIF-iso4E and the S6 ribosomal protein, was also found after auxin stimulation of maize axes. A positive correlation was established between the levels of S6 ribosomal protein phosphorylation and the S6 ribosomal protein transcript recruitment into polysomes by means of okadaic acid or heat shock application to maize axes. These data indicate that auxin stimulates S6 ribosomal protein phosphorylation on maize ribosomes, concomitant to the recruitment of specific mRNAs (5'TOP-like mRNAs) into polysomes for translation. It is proposed that by this mechanism auxin regulate the synthesis of specific proteins in maize tissues.

Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3'-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.

Hace 150 anos se publico la teoria de Darwin sobre la evolucion que dice: "Las caracteristicas de un organismo son hereditarias y sujetas a cambios al azar; los cambios que permiten al organismo mejorar sus opciones de supervivencia, se conservaran en las futuras generaciones". El Premio Nobel en Quimica 2009 fue otorgado a tres cientificos que aportaron la ultima pieza requerida para conocer como funciona en realidad la teoria de Darwin a nivel atomico. El primer premio Nobel en este sentido fue uno de los mas famosos en la historia: el otorgado en 1962 a Francis Crick, James Watson y Maurice Wilkins por el modelo atomico de la doble helice de ADN. El segundo fue el otorgado en el 2006 a Roger Kornberg por la estructura de la ARN polimerasa que copia la informacion de ADN a ARN. El tercero, otorgado este ano a Ada Yonath, Thomas Steitz y Venkatraman Ramakrishnan por la estructura cristalina de alta resolucion del ribosoma – una de las maquinarias celulares mas complejas – que completa el camino de la informacion genetica, permitiendonos conocer como el lenguaje de ADN/ARN se convierte en proteinas que garantizan el correcto funcionamiento celular. En los anos sesenta se sucedieron varios descubrimientos clave para determinar que la informacion genetica codificada a nivel de ADN es primero copiada a ARN mensajero (ARNm) y luego exportada fuera del nucleo. En el citoplasma, los ribosomas y los ARN de transferencia (ARNt) decodifican la informacion a secuencia de aminoacidos formando una proteina. El codigo genetico fue completamente elucidado y solo restaba conocer como funcionan a nivel quimico estas moleculas para garantizar un proceso tan asombrosamente preciso, rapido y eficiente que ademas se ha conservado desde las bacterias hasta los humanos

Mechanisms for regulation of gene expression at the translational level have been reported at specific developmental stages in eukaryotes. Control of eukaryotic initiation factor (eIF) 4E availability by insulin/growth factors constitutes a main point of translational regulation. The aim of the present research was to understand the regulatory mechanism(s) behind the differential expression of two main 4E factors present in maize embryonic axes during germination. De novo synthesis of eIFiso4E initiates earlier and is faster than that of eIF4E in maize axes. Insulin addition to maize axes stimulated de novo synthesis of the eIFiso4E protein, but not that of eIF4E. Specific recruitment of the eIFiso4E transcript into polysomes was observed in these axes after insulin stimulation. Inhibitors of the insulin signal-transduction pathway, wortmannin and rapamycin, reversed the insulin effect. In vitro translation of maize poly(A)+ RNAs by S6 ribosomal protein (rp)-phosphorylated ribosomes demonstrated a strong increase in eIFiso4E synthesis, as compared with its translation by S6 rp-non-phosphorylated ribosomes. Other mRNAs from the poly(A)+ RNA set, including the eIF4E mRNA, did not show differential translation with regard to the S6-phosphorylated status of the ribosomes. The overall results indicate that eIFiso4E, but not eIF4E, cell content is regulated by de novo synthesis in maize axes during germination, most probably by specific mRNA recruitment into polysomes via a signal-transduction pathway involving S6 rp phosphorylation.

Messenger RNA translation is a tightly regulated event in maize axes during germination. To obtain a deeper understanding of this process, the present research was centered on the expression regulation of the translation initiation factor elF-4E within this period. Two maize eIF-4E isoforms were isolated by 7 mGTP-Sepharose affinity chromatography. The eIF-iso4E protein content remained at a constant level in the axes during the first 24 h of maize germination, whereas the elF-4E level increased after 12 h of germination as indicated by western blot analysis. Fast in vivo eIF-iso4E, but not eIF-4E, de novo synthesis was found within 6-12 h of germination. Northern blot analysis of total RNA from ungerminated axes with cDNAs encoding either -iso4E or -4E indicated that both transcripts were present in the stored mRNA pool of maize axes. Transcription for any of these mRNAs was not detected before 12 h of germination. Northern blots of polysomal RNAs indicated that the eIF-iso4E stored transcript is selectively recruited into polysomes for translation as early as 6 h of germination, whereas this does not occur for the eIF-4E transcript. The above data demonstrate differential 4E isoform expression regulation during maize germination. Expression of the eIF-iso4E protein appears to be translationally controlled, whereas the expression of the eIF-4E protein might be regulated at the transcriptional level.

This article focuses on the effect that S6 ribosomal protein phosphorylation might have in regulating mRNA translation. Maize axes of either 4 or 14 h of germination were pulse-labelled for 1 h with [32P]-orthophosphate. Analysis of their ribosomal proteins by gel electrophoresis and autoradiography showed distinctive levels of S6 ribosomal protein phosphorylation for both ribosomal sets. Axes at these two stages of germination were treated with alpha-amanitin to ensure transcription inhibition and pulse-labelling with [35S]-methionine. The [35S]-proteins, resulting from stored mRNA translation, when analysed by 2-D-gel electrophoresis and fluorography revealed distinctive [35S]-protein patterns. In vitro translation of stored mRNA on ribosomes from either 4 or 14 h germinated-maize axes produced different [35S]-protein patterns. Further, addition of 7methyl-GTP-Sepharose to the translation system showed differential cap-dependent protein synthesis inhibition depending on the set of ribosomes tested. It is concluded that translation of stored mRNA in germinating maize axes is at least partially regulated by a mechanism that involves S6 ribosomal protein phosphorylation.

Ribosome biogenesis is a pre-requisite for cell growth and proliferation; it is however, a highly regulated process that consumes a great quantity of energy. It requires the coordinated production of rRNA, ribosomal proteins and non-ribosomal factors which participate in the processing and mobilization of the new ribosomes. Ribosome biogenesis has been studied in yeast and animals; however, there is little information about this process in plants. The objective of the present work was to study ribosome biogenesis in maize seeds during germination, a stage characterized for its fast growth, and the effect of insulin in this process. Insulin has been reported to accelerate germination and to induce seedling growth. It was observed that among the first events reactivated just after 3 h of imbibition are the rDNA transcription and the pre-rRNA processing and that insulin stimulates both of them (40-230%). The transcript of nucleolin, a protein which regulates rDNA transcription and pre-rRNA processing, is among the messages stored in quiescent dry seeds and it is mobilized into the polysomal fraction during the first hours of imbibition (6 h). In contrast, de novo ribosomal protein synthesis was low during the first hours of imbibition (3 and 6 h) increasing by 60 times in later stages (24 h). Insulin increased this synthesis (75%) at 24 h of imbibition; however, not all ribosomal proteins were similarly regulated. In this regard, an increase in RPS6 and RPL7 protein levels was observed, whereas RPL3 protein levels did not change even though its transcription was induced. Results show that ribosome biogenesis in the first stages of imbibition is carried out with newly synthesized rRNA and ribosomal proteins translated from stored mRNA.

This work describes the identification of components in the Cap-binding complexes in non-germinated and 24-h-imbibed seeds using mass spectrometry. This approach revealed new components particularly present in the non-germinated seed. Among these, two heat shock proteins, HSP101 and HSP70, were detected as well as several proteins involved in carbohydrate metabolism. Between the new components of maize Cap-binding complexes, several proteins contain a motif that identifies them as potential direct interactors with eIF4E or eIF(iso)4E.Together with the major abundance of eIF(iso)4E at this developmental stage, our findings indicate clear differences between the translation initiation complexes that are available for protein synthesis right upon water imibition and those that are present once germination has been completed.

C. elegans represents a favorable system to study the extraordinarily complicated process of eukaryotic protein synthesis, which involves over 100 RNAs and over 200 polypeptides just for the core machinery. Initial research in protein synthesis relied on fractionated mammalian and plant systems, but in the mid-1970s, the powerful genetics of Saccharomyces cerevisiae began to yield new insights for translation in all eukaryotes. C. elegans has many features of higher eukaryotes that are not shared by yeast. This allows protein synthesis researchers to combine biochemistry, cell biology, developmental biology, genetics, and genomics to study regulation of gene expression at the translational level. Most components of the core translational machinery have been identified in C. elegans, including rRNAs, 5S RNA, tRNAs, ribosomal proteins, and aminoacyl tRNA synthetases. C. elegans has amino acid sequence homologs for 56 of the known initiation, elongation, and release factor polypeptides, but few of these have been isolated, functionally identified, or studied at the biochemical level. Similarly, C. elegans has homologs for 22 components of the major signal transduction pathways implicated in control of protein synthesis. The translational efficiency of individual mRNAs relies on cis-regulatory elements that include either a 7-methylguanosine- or 2,2,7-trimethylguanosine-containing cap, the 5'-terminal spliced leader, sequence elements in the 3'-untranslated regions, and the 3'-terminal poly(A) tract. Several key developmental pathways in C. elegans are predominantly governed by translational mechanisms. Some evidence has been presented that well described regulatory mechanisms in other organisms, including covalent modification of translation factors, sequestration of translation factors, and mRNA-specific changes in poly(A) length, also occur in C. elegans. The most interesting unexplored questions may involve changes in the translation of individual mRNAs during development, in response to physiological changes, or after genetic manipulations. Given the highly developed state of C. elegans genomics, it can be expected that future application of computational tools, including data visualization, will help detect new instances of translational control.

The translational initiation factor eIF4E binds to the m(7)G-containing cap of mRNA and participates in recruitment of mRNA to ribosomes for protein synthesis. eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol. Multiple eIF4E family members have been identified in a wide range of organisms that includes plants, flies, mammals, frogs, birds, nematodes, fish, and various protists. This chapter reviews methods that have been applied to learn the biochemical properties and physiological functions that differentiate eIF4E family members within a given organism. Much has been learned to date about approaches to discover new eIF4E family members, their in vitro properties (cap binding, stimulation of cell-free translation systems), tissue and developmental expression patterns, protein-binding partners, and their effects on the translation or repression of specific subsets of mRNA. Despite these advances, new eIF4E family members continue to be found and new physiological roles discovered.

The translational initiation factor eIF4E binds to the m(7)G-containing cap of mRNA and participates in recruitment of mRNA to ribosomes for protein synthesis. eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol. Multiple eIF4E family members have been identified in a wide range of organisms that includes plants, flies, mammals, frogs, birds, nematodes, fish, and various protists. This chapter reviews methods that have been applied to learn the biochemical properties and physiological functions that differentiate eIF4E family members within a given organism. Much has been learned to date about approaches to discover new eIF4E family members, their in vitro properties (cap binding, stimulation of cell-free translation systems), tissue and developmental expression patterns, protein-binding partners, and their effects on the translation or repression of specific subsets of mRNA. Despite these advances, new eIF4E family members continue to be found and new physiological roles discovered.

The relationships between genes in neighboring clusters in a self-organizing map (SOM) and properties attributed to them are sometimes difficult to discern, especially when heterogeneous datasets are used. We report a novel approach to identify correlations between heterogeneous datasets. One dataset, derived from microarray analysis of polysomal distribution, contained changes in the translational efficiency of Caenorhabditis elegans mRNAs resulting from loss of specific eIF4E isoform. The other dataset contained expression patterns of mRNAs across all developmental stages. Two algorithms were applied to these datasets: a classical scatter plot and an SOM. The outputs were linked using a two-dimensional color scale. This revealed that an mRNA’s eIF4E-dependent translational efficiency is strongly dependent on its expression during development. This correlation was not detectable with a traditional one-dimensional color scale.