A phosphorylation-deficient ribosomal protein eS6 is largely functional in , rescuing mutant defects from global translation and gene expression to photosynthesis and growth.

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from , which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions.

Prioritization of the concepts and skills in quantitative education for graduate students in biomedical science.

Substantial guidance is available on undergraduate quantitative training for biologists, including reports focused on biomedical science. Far less attention has been paid to the graduate curriculum and the particular challenges of the diversity of specialization within the life sciences. We propose an innovative approach to quantitative education that goes beyond recommendations of a course or set of courses or activities, derived from analysis of the expectations for students in particular programs.

A Miniature Sucrose Gradient for Polysome Profiling.

-10 mL sucrose gradient onto which 0.5-1 mL of cell extract is layered and centrifuged at high speed for 3-4 h in a floor-model ultracentrifuge. After centrifugation, the gradient solution is passed through an absorbance recorder to generate a polysome profile.

Modulation of GCN2 activity under excess light stress by osmoprotectants and amino acids.

The protein kinase GCN2 (General Control Nonderepressible2) and its phosphorylation target, the eukaryotic translation initiation factor (eIF)2α represent the core module of the plant's integrated stress response, a signaling pathway widely conserved in eukaryotes that can rapidly regulate translation in response to stressful conditions. Recent findings indicate that the GCN2 protein operates under the command of reactive oxygen species (ROS) emanating from the chloroplast under a variety of abiotic stresses such as excess light. To get deeper insights into the mechanism of GCN2 activation under excess light, we assessed the role of amino acids in view of the classic function of GCN2 as a sensor of amino acid status.

Review: Emerging roles of the signaling network of the protein kinase GCN2 in the plant stress response.

The pan-eukaryotic protein kinase GCN2 (General Control Nonderepressible2) regulates the translation of mRNAs in response to external and metabolic conditions. Although GCN2 and its substrate, translation initiation factor 2 (eIF2) α, and several partner proteins are substantially conserved in plants, this kinase has assumed novel functions in plants, including in innate immunity and retrograde signaling between the chloroplast and cytosol. How exactly some of the biochemical paradigms of the GCN2 system have diverged in the green plant lineage is only partially resolved.

Early Detection of Daylengths with a Feedforward Circuit Coregulated by Circadian and Diurnal Cycles.

Light-entrained circadian clocks confer rhythmic dynamics of cellular and molecular activities to animals and plants. These intrinsic clocks allow stable anticipations to light-dark (diel) cycles. Many genes in the model plant Arabidopsis thaliana are regulated by diel cycles via pathways independent of the clock, suggesting that the integration of circadian and light signals is important for the fitness of plants.

Light-Dependent Activation of the GCN2 Kinase Under Cold and Salt Stress Is Mediated by the Photosynthetic Status of the Chloroplast.

Regulation of cytosolic mRNA translation is a key node for rapid adaptation to environmental stress conditions. In yeast and animals, phosphorylation of the α-subunit of eukaryotic translation initiation factor eIF2 is the most thoroughly characterized event for regulating global translation under stress. In plants, the GCN2 kinase () is the only known kinase for eIF2α.

Translational gene regulation in plants: A green new deal.

The molecular machinery for protein synthesis is profoundly similar between plants and other eukaryotes. Mechanisms of translational gene regulation are embedded into the broader network of RNA-level processes including RNA quality control and RNA turnover. However, over eons of their separate history, plants acquired new components, dropped others, and generally evolved an alternate way of making the parts list of protein synthesis work.

Light Activates the Translational Regulatory Kinase GCN2 via Reactive Oxygen Species Emanating from the Chloroplast.

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of the translation initiation factor eIF2α anchors a reversible regulatory switch that represses cytosolic translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2α serine 56 in Arabidopsis ().

ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem.

The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis () are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions.

Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals.

The translation of mRNA into protein is tightly regulated by the light environment as well as by the circadian clock. Although changes in translational efficiency have been well documented at the level of mRNA-ribosome loading, the underlying mechanisms are unclear. The reversible phosphorylation of RIBOSOMAL PROTEIN OF THE SMALL SUBUNIT 6 (RPS6) has been known for 40 years, but the biochemical significance of this event remains unclear to this day.

Graduate Training at the Interface of Computational and Experimental Biology: An Outcome Report from a Partnership of Volunteers between a University and a National Laboratory.

Leading voices in the biological sciences have called for a transformation in graduate education leading to the PhD degree. One area commonly singled out for growth and innovation is cross-training in computational science. In 1998, the University of Tennessee (UT) founded an intercollegiate graduate program called the UT-ORNL Graduate School of Genome Science and Technology in partnership with the nearby Oak Ridge National Laboratory.

What makes ribosomes tick?

In most organisms, gene expression over the course of the day is under the control of the circadian clock. The canonical clock operates as a gene expression circuit that is controlled at the level of transcription, and transcriptional control is also a major clock output. However, rhythmic transcription cannot explain all the observed rhythms in protein accumulation.

Meeting report: processing, translation, decay - three ways to keep RNA sizzling.

This meeting report highlights key trends that emerged from a conference entitled Post-Transcriptional Gene Regulation in Plants, which was held 14-15 July 2016, as a satellite meeting of the annual meeting of the American Society of Plant Biologists in Austin, Texas. The molecular biology of RNA is emerging as an integral part of the framework for plants' responses to environmental challenges such as drought and heat, hypoxia, nutrient deprivation, light and pathogens. Moreover, the conference illustrated how a multitude of customized and pioneering omics-related technologies are being applied, more and more often in combination, to describe and dissect the complexities of gene expression at the post-transcriptional level.

The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading.

Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes.

Analysis of mRNA translation states in Arabidopsis over the diurnal cycle by polysome microarray.

Gene regulation at the level of translation occurs in response to environmental perturbation and is increasingly recognized as a factor affecting plant development. Despite extensive knowledge of transcriptional control, very little is known about translational regulation of genes in response to the daily light/dark cycles. Here we describe the experimental layout designed to address how the translation states of genes change at various times during a diurnal cycle in Arabidopsis thaliana seedlings.

Translational control of Arabidopsis meristem stability and organogenesis by the eukaryotic translation factor eIF3h.

Essentially all aboveground plant tissues develop from the stem cells in the primary shoot apical meristem. Proliferation of the stem cell population in the Arabidopsis shoot apical meristem is tightly controlled by a feedback loop formed primarily by the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA ligand-receptor system. In this study, it is shown that mutation of a translation initiation factor, eIF3h, causes a tendency to develop a strikingly enlarged shoot apical meristem with elevated and ectopic expression of WUS and CLAVATA3 (CLV3).

The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant.

Background: Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1.

Results: The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins.

Regulation of plant translation by upstream open reading frames.

We review the evidence that upstream open reading frames (uORFs) function as RNA sequence elements for post-transcriptional control of gene expression, specifically translation. uORFs are highly abundant in the genomes of angiosperms. Their negative effect on translation is often attenuated by ribosomal translation reinitiation, a process whose molecular biochemistry is still being investigated.

Translational Regulation of Cytoplasmic mRNAs.

Translation of the coding potential of a messenger RNA into a protein molecule is a fundamental process in all living cells and consumes a large fraction of metabolites and energy resources in growing cells. Moreover, translation has emerged as an important control point in the regulation of gene expression. At the level of gene regulation, translational control is utilized to support the specific life histories of plants, in particular their responses to the abiotic environment and to metabolites.

Arabidopsis BPG2: a phytochrome-regulated gene whose protein product binds to plastid ribosomal RNAs.

BPG2 (Brz-insensitive pale green 2) is a dark-repressible and light-inducible gene that is required for the greening process in Arabidopsis. Light pulse experiments suggested that light-regulated gene expression of BPG2 is mediated by phytochrome. The T-DNA insertion mutant bpg2-2 exhibited a reduced level of chlorophyll and carotenoid pigmentation in the plastids.

Fluorescence-tagged transgenic lines reveal genetic defects in pollen growth--application to the eIF3 complex.

Background: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.

Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24.

Background: Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs).

The h subunit of eIF3 promotes reinitiation competence during translation of mRNAs harboring upstream open reading frames.

Upstream open reading frames (uORFs) are protein coding elements in the 5' leader of messenger RNAs. uORFs generally inhibit translation of the main ORF because ribosomes that perform translation elongation suffer either permanent or conditional loss of reinitiation competence. After conditional loss, reinitiation competence may be regained by, at the minimum, reacquisition of a fresh methionyl-tRNA.

YABBYs and the transcriptional corepressors LEUNIG and LEUNIG_HOMOLOG maintain leaf polarity and meristem activity in Arabidopsis.

In Arabidopsis thaliana, FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) encode YABBY domain proteins that regulate abaxial patterning, growth of lateral organs, and inflorescence phyllotaxy. In this study, we show that YABs physically interact with components of a transcriptional repressor complex that include LEUNIG (LUG), LEUNIG_HOMOLOG (LUH), the LUG-associated coregulator SEUSS, and related SEUSS-LIKE proteins. Consistent with the formation of a LUG-YAB complex, we find that lug mutants enhance the polarity and growth defects of fil yab3 mutant leaves and that this enhancement is due to a loss of LUG activity from the abaxial domain.