Ubiquitin-mediated mechanisms of translational control.

mRNAs translation to proteins constitutes an important step of cellular gene expression that is highly regulated in response to different extracellular stimuli and stress situations. The fine control of protein synthesis is carried out both qualitatively and quantitatively, depending on the cellular demand at each moment. Post-translational modifications, in turn regulated by intracellular signaling pathways, play a key role in translation regulation. Among them, ubiquitination, whose role is becoming increasingly important in the control of translation, determines a correct balance between protein synthesis and degradation. In this review we focus on the role of ubiquitination (both degradative K48-linkage type and non-degradative K63-linkage type and monoubiquitination) in eukaryotic translation, both at the pre-translational level during the biogenesis/degradation of the components of translational machinery as well as at the co-translational level under stressful conditions. We also discuss other ubiquitin-dependent regulatory mechanisms of mRNA protection and resumption of translation after stress removal, where the ubiquitination of ribosomal proteins and associated regulatory proteins play an important role in the global rhythm of translation.

An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates.

Posttranslational modifications (PTMs), particularly phosphorylation, play a pivotal role in expanding the complexity of the proteome and regulating diverse cellular processes. In this study, we present an efficient Escherichia coli phosphorylation system designed to streamline the evaluation of potential substrates for Arabidopsis thaliana plant kinases, although the technology is amenable to any. The methodology involves the use of IPTG-inducible vectors for co-expressing kinases and substrates, eliminating the need for radioactive isotopes and prior protein purification. We validated the system's efficacy by assessing the phosphorylation of well-established substrates of the plant kinase SnRK1, including the rat ACETYL-COA CARBOXYLASE 1 (ACC1) and FYVE1/FREE1 proteins. The results demonstrated the specificity and reliability of the system in studying kinase-substrate interactions. Furthermore, we applied the system to investigate the phosphorylation cascade involving the A. thaliana MKK3-MPK2 kinase module. The activation of MPK2 by MKK3 was demonstrated to phosphorylate the Myelin Basic Protein (MBP), confirming the system's ability to unravel sequential enzymatic steps in phosphorylation cascades. Overall, this E. coli phosphorylation system offers a rapid, cost-effective, and reliable approach for screening potential kinase substrates, presenting a valuable tool to complement the current portfolio of molecular techniques for advancing our understanding of kinase functions and their roles in cellular signaling pathways.

Eukaryotic Initiation Factor 5A2 localizes to actively translating ribosomes to promote cancer cell protrusions and invasive capacity.

BACKGROUND: Eukaryotic Initiation Factor 5A (eIF-5A), an essential translation factor, is post-translationally activated by the polyamine spermidine. Two human genes encode eIF-5A, being eIF5-A1 constitutively expressed whereas eIF5-A2 is frequently found overexpressed in human tumours. The contribution of both isoforms with regard to cellular proliferation and invasion in non-small cell lung cancer remains to be characterized. METHODS: We have evaluated the use of eIF-5A2 gene as prognosis marker in lung adenocarcinoma (LUAD) patients and validated in immunocompromised mice. We have used cell migration and cell proliferation assays in LUAD lines after silencing each eIF-5A isoform to monitor their contribution to both phenotypes. Cytoskeleton alterations were analysed in the same cells by rhodamine-phalloidin staining and fluorescence microscopy. Polysome profiles were used to monitor the effect of eIF-5A2 overexpression on translation. Western blotting was used to study the levels of eIF-5A2 client proteins involved in migration upon TGFB1 stimulation. Finally, we have co-localized eIF-5A2 with puromycin to visualize the subcellular pattern of actively translating ribosomes. RESULTS: We describe the differential functions of both eIF-5A isoforms, to show that eIF5-A2 properties on cell proliferation and migration are coincident with its features as a poor prognosis marker. Silencing of eIF-5A2 leads to more dramatic consequences of cellular proliferation and migration compared to eIF-5A1. Overexpression of eIF-5A2 leads to enhanced global translation. We also show that TGFß signalling enhances the expression and activity of eIF-5A2 which promotes the translation of polyproline rich proteins involved in cytoskeleton and motility features as it is the case of Fibronectin, SNAI1, Ezrin and FHOD1. With the use of puromycin labelling we have co-localized active ribosomes with eIF-5A2 not only in cytosol but also in areas of cellular protrusion. We have shown the bulk invasive capacity of cells overexpressing eIF-5A2 in mice. CONCLUSIONS: We propose the existence of a coordinated temporal and positional interaction between TFGB and eIF-5A2 pathways to promote cell migration in NSCLC. We suggest that the co-localization of actively translating ribosomes with hypusinated eIF-5A2 facilitates the translation of key proteins not only in the cytosol but also in areas of cellular protrusion. Video Abstract.

Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth.

Plant reproduction is one key biological process that is very sensitive to heat stress and, as a result, enhanced global warming becomes a serious threat to agriculture. In this work, we have studied the effects of heat on germinated pollen of Arabidopsis thaliana both at the transcriptional and translational level. We have used a high-resolution ribosome profiling technology to provide a comprehensive study of the transcriptome and the translatome of germinated pollen at permissive and restrictive temperatures. We have found significant down-regulation of key membrane transporters required for pollen tube growth by heat, thus uncovering heat-sensitive targets. A subset of the heat-repressed transporters showed coordinated up-regulation with canonical heat-shock genes at permissive conditions. We also found specific regulations at the translational level and we have uncovered the presence of ribosomes on sequences annotated as non-coding. Our results demonstrate that heat impacts mostly on membrane transporters thus explaining the deleterious effects of heat stress on pollen growth. The specific regulations at the translational level and the presence of ribosomes on non-coding RNAs highlights novel regulatory aspects on plant fertilization.

Citrus exocortis viroid causes ribosomal stress in tomato plants.

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.

Performance of the eclipse monitor unit objective tool utilizing volumetric modulated arc therapy for rectal cancer.

AIM: To assess the performance of the monitor unit (MU) Objective tool in Eclipse treatment planning system (TPS) utilizing volumetric modulated arc therapy (VMAT) for rectal cancer. BACKGROUND: Eclipse VMAT planning module includes a tool to control the number of MUs delivered: the MU Objective tool. This tool could be utilized to reduce the total number of MUs in rectal cancer treatments. MATERIALS AND METHODS: 20 rectal cancer patients were retrospectively studied using VMAT and the MU Objective tool. The baseline plan for each patient was selected as the one with no usage of the MU Objective tool. The number of MUs of this plan was set to be the reference number of MUs (MUref). Five plans were re-optimized for each patient only varying the Max MU parameter. The selected values were 30%, 60%, 90%, 120% and 150% of MUref for each patient. Differences with respect to the baseline plan were evaluated regarding MU number and parameters for PTVs coverage evaluation, PTVs homogeneity and OARs doses assessment. A two-tailed, paired-samples t-test was used to quantify these differences. RESULTS: Average relative differences in MU number obtained was 10% for Max MU values of 30% and 60% of MUref, respectively (p < 0.03). PTVs coverage and homogeneity were not compromised and discrepancies obtained with respect to baseline plans were not significant. Furthermore, maximum OARs doses deviations were also not significant. CONCLUSIONS: A 10% reduction in the MU number could be obtained without an alteration of PTV coverage and OARs doses for rectal cancer.

Management of interruptions in radiotherapy treatments: Adaptive implementation in high workload sites.

Owing to predictable or unpredictable causes, interruptions may arise during therapy. On average, the extension of fractionated radiotherapy treatments is prone to be delayed by several weeks and interruptions can come up extending overall treatment time (OTT). Clonogenic cells of aggressive tumors might benefit from this situation, modifying local control (LC). Preserving treatment quality in radiotherapy is an essential issue for the treatment outcome, and our institution is increasingly concerned about this line of work. Establishing some objective criteria to schedule patients that have suffered interruptions along their treatments is of capital importance and not a trivial issue. Publications strongly encourage departments to minimize the effect of lag periods during treatments. Therefore, in July 2017, our facility implemented the so called 'Protocol to Manage Interruptions in Radiotherapy', based on a scoring system for patient categorization that considers not only histology but also associated comorbidity and sequence of the therapy.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

Regulation of Translation Initiation under Biotic and Abiotic Stresses.

Plants have developed versatile strategies to deal with the great variety of challenging conditions they are exposed to. Among them, the regulation of translation is a common target to finely modulate gene expression both under biotic and abiotic stress situations. Upon environmental challenges, translation is regulated to reduce the consumption of energy and to selectively synthesize proteins involved in the proper establishment of the tolerance response. In the case of viral infections, the situation is more complex, as viruses have evolved unconventional mechanisms to regulate translation in order to ensure the production of the viral encoded proteins using the plant machinery. Although the final purpose is different, in some cases, both plants and viruses share common mechanisms to modulate translation. In others, the mechanisms leading to the control of translation are viral- or stress-specific. In this paper, we review the different mechanisms involved in the regulation of translation initiation under virus infection and under environmental stress in plants. In addition, we describe the main features within the viral RNAs and the cellular mRNAs that promote their selective translation in plants undergoing biotic and abiotic stress situations.

Characterization of Arabidopsis Post-Glycosylphosphatidylinositol Attachment to Proteins Phospholipase 3 Like Genes.

Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1/PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus.

Extremophilic bacteria restrict the growth of Macrophomina phaseolina by combined secretion of polyamines and lytic enzymes.

Extremophilic microorganisms were screened as biocontrol agents against two strains of Macrophomina phaseolina (Mp02 and 06). Stenotrophomonas sp. AG3 and Exiguobacterium sp. S58 exhibited a potential in vitro antifungal effect on Mp02 growth, corresponding to 52.2% and 40.7% inhibition, respectively. This effect was confirmed by scanning electron microscopy, where images revealed marked morphological alterations in fungus hyphae. The bacteria were found to secrete lytic enzymes and polyamines. Exiguobacterium sp. S56a was the only strain able to reduce the growth of the two strains of M. phaseolina through their supernatant. Antifungal supernatant activity was correlated with the ability of bacteria to synthesize and excrete putrescine, and the exogenous application of this polyamine to the medium phenocopied the bacterial antifungal effects. We propose that the combined secretion of putrescine, spermidine, and lytic enzymes by extremophilic microorganism predispose these microorganisms to reduce the disease severity occasioned by M. phaseolina in soybean seedlings.

Characterization of novel pollen-expressed transcripts reveals their potential roles in pollen heat stress response in Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.

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

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.

Dosimetric Comparison of Four Volumetric-Modulated Arc Therapy Beam Arrangements Utilized for Hippocampal-Sparing Whole-Brain Radiation Therapy.

PURPOSE: In the present study, the performance of four VMAT beam arrangements used for hippocampal-sparing whole-brain radiation therapy is addressed. MATERIAL AND METHODS: Data corresponding to 20 patients were utilized so as to generate plans for every beam configuration. A preliminary study was conducted to assess the optimal distance between optimization structures (PTVx) and hippocampi. V25, V30, D50%, D2%, D98%, homogeneity index (HI) and Paddick conformity factor (CF) were evaluated for PTV. D100% and Dmax were considered for hippocampi. All plans were required to perform at least as recommended in RTOG 0933 trial regarding organs at risk (OAR) sparing and PTV objectives. RESULTS: Considerable hippocampi sparing alongside with a reasonably low decrease in PTV coverage was achieved using a 7 mm distance between hippocampi and PTV optimization structure. Beam setup 3 (comprised of two full arcs with 0° couch angle and two half arcs with 90° couch angle) achieved the best PTV coverage, HI and CF, while it performed the second-best sparing in hippocampi and lenses. Moreover, beam setup 3 was the second-fastest treatment, although it resulted in the highest number of delivered MU among all beam setups. Beam setup 1 (comprised of two full arcs with no couch angles) was the fastest and it delivered a significantly less amount of monitor units compared with the other beam setups evaluated. Furthermore, a higher robustness was obtained by using no couch angles. Although beam setup 1 was the least optimal considering OAR sparing, it still performed better than required in the RTOG 0933 trial. CONCLUSIONS: Overall, beam setup 3 was considered to be the best. It is worth mentioning that, apart from our results, the election of one of these beam arrangements might be dependent on the amount of patient workload at a specific institution.

Polyamines interfere with protein ubiquitylation and cause depletion of intracellular amino acids: a possible mechanism for cell growth inhibition.

Spermidine is a polyamine present in eukaryotes with essential functions in protein synthesis. At high concentrations spermidine and norspermidine inhibit growth by unknown mechanisms. Transcriptomic analysis of the effect of norspermidine on the plant Arabidopsis thaliana indicates upregulation of the response to heat stress and denatured proteins. Accordingly, these polyamines inhibit protein ubiquitylation, both in vivo (in yeast, Arabidopsis, and human Hela cells) and in vitro (with recombinant ubiquitin ligase). This interferes with protein degradation by the proteasome, a situation known to deplete cells of amino acids. Norspermidine treatment of yeast cells induces amino acid depletion, and supplementation of media with amino acids counteracts growth inhibition and cellular amino acid depletion but not inhibition of protein polyubiquitylation.

Involvement of polyamines in plant response to abiotic stress.

Environmental stresses are the major cause of crop loss worldwide. Polyamines are involved in plant stress responses. However, the precise role(s) of polyamine metabolism in these processes remain ill-defined. Transgenic approaches demonstrate that polyamines play essential roles in stress tolerance and open up the possibility to exploit this strategy to improve plant tolerance to multiple environmental stresses. The use of Arabidopsis as a model plant enables us to carry out global expression studies of the polyamine metabolic genes under different stress conditions, as well as genome-wide expression analyses of insertional-mutants and plants over-expressing these genes. These studies are essential to dissect the polyamine mechanism of action in order to design new strategies to increase plant survival in adverse environments.

Quantitation of Protein Translation Rate In Vivo with Bioorthogonal Click-Chemistry.

The development of novel bioorthogonal reactives that can be used to tag biomolecules in vivo has revolutionized the studies of cellular and molecular biology. Among those novel reactive substances, amino acid analogs can be used to label nascent proteins, thus opening new avenues for measuring protein translation rates in vivo with a limited manipulation of the sample. Here, we describe the use of Click-chemistry to tag and separate newly synthesized proteins in mammalian cells that can be used, coupled with western analysis, to estimate the translation rate of any protein of interest.

Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development.

One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of eIF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development.