Nathaniel P. Hoyle

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

Glucose depletion inhibits translation initiation via eIF4A loss and subsequent 48S preinitiation complex accumulation, while the pentose phosphate pathway is coordinately up-regulated.

The mechanism and consequences of the translational inhibition caused by glucose depletion in yeast are characterized. eIF4A is lost from the preinitiation complex, and the pentose phosphate pathway is translationally up-regulated, allowing an efficient transition to the new conditions.

Daily magnesium fluxes regulate cellular timekeeping and energy balance

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg2+]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg2+ as a cofactor for ATP, a functional consequence of [Mg2+]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg2+ availability has potential to impact upon many of the cell’s more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg2+]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control.

The eukaryotic translation initiation factor 2B (eIF2B) provides a fundamental controlled point in the pathway of protein synthesis. eIF2B is the heteropentameric guanine nucleotide exchange factor that converts eIF2, from an inactive guanosine diphosphate–bound complex to eIF2-guanosine triphosphate. This reaction is controlled in response to a variety of cellular stresses to allow the rapid reprogramming of cellular gene expression. Here we demonstrate that in contrast to other translation initiation factors, eIF2B and eIF2 colocalize to a specific cytoplasmic locus. The dynamic nature of this locus is revealed through fluorescence recovery after photobleaching analysis. Indeed eIF2 shuttles into these foci whereas eIF2B remains largely resident. Three different strategies to decrease the guanine nucleotide exchange function of eIF2B all inhibit eIF2 shuttling into the foci. These results implicate a defined cytoplasmic center of eIF2B in the exchange of guanine nucleotides on the eIF2 translation initiation factor. A focused core of eIF2B guanine nucleotide exchange might allow either greater activity or control of this elementary conserved step in the translation pathway.

Transcript processing and export kinetics are rate-limiting steps in expressing vertebrate segmentation clock genes

Significance This paper describes in vivo measurements of the kinetics of transcript processing and export for endogenous genes in mouse and chick embryos. It shows that transcript export is unexpectedly slow, even slower than splicing, and relates its finding to rate-limiting steps that would contribute to the molecular oscillator that drives segmentation in vertebrate embryos. It also relates them to interspecies differences in clock period. Sequential production of body segments in vertebrate embryos is regulated by a molecular oscillator (the segmentation clock) that drives cyclic transcription of genes involved in positioning intersegmental boundaries. Mathematical modeling indicates that the period of the clock depends on the total delay kinetics of a negative feedback circuit, including those associated with the synthesis of transcripts encoding clock components [Lewis J (2003) Curr Biol 13(16):1398–1408]. Here, we measure expression delays for three transcripts [Lunatic fringe, Hes7/her1, and Notch-regulated-ankyrin-repeat-protein (Nrarp)], that cycle during segmentation in the zebrafish, chick, and mouse, and provide in vivo measurements of endogenous splicing and export kinetics. We show that mRNA splicing and export are much slower than transcript elongation, with the longest delay (about 16 min in the mouse) being due to mRNA export. We conclude that the kinetics of mRNA and protein production and destruction can account for much of the clock period, and provide strong support for delayed autorepression as the underlying mechanism of the segmentation clock.

Oxidation–Reduction Cycles of Peroxiredoxin Proteins and Nontranscriptional Aspects of Timekeeping

The circadian clock allows organisms to accurately predict the earth’s rotation and modify their behavior as a result. Genetic analyses in a variety of organisms have defined a mechanism based largely on gene expression feedback loops. However, as we delve more deeply into the mechanisms of circadian timekeeping, we are discovering that post-translational mechanisms play a key role in defining the character of the clock. We are also discovering that these modifications are inextricably linked to cellular metabolism, including redox homeostasis. A robust circadian oscillation in the redox status of the peroxiredoxins (a major class of cellular antioxidants) was recently shown to be remarkably conserved from archaea and cyanobacteria all the way to plants and animals. Furthermore, recent findings indicate that cellular redox status is coupled not only to canonical circadian gene expression pathways but also to a noncanonical transcript-independent circadian clock. The redox rhythms observed in peroxiredoxins in the absence of canonical clock mechanisms may hint at the nature of this new and hitherto unknown aspect of circadian timekeeping.

Granules Harboring Translationally Active mRNAs Provide a Platform for P-Body Formation following Stress

Summary The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

Circadian clocks, brain function, and development

Circadian clocks are temporal interfaces that organize biological systems and behavior to dynamic external environments. Components of the molecular clock are expressed throughout the brain and are centrally poised to play an important role in brain function. This paper focuses on key issues concerning the relationship among circadian clocks, brain function, and development, and discusses three topic areas: (1) sleep and its relationship to the circadian system; (2) systems development and psychopathology (spanning the prenatal period through late life); and (3) circadian factors and their application to neuropsychiatric disorders. We also explore circadian genetics and psychopathology and the selective pressures on the evolution of clocks. Last, a lively debate is presented on whether circadian factors are central to mood disorders. Emerging from research on circadian rhythms is a model of the interaction among genes, sleep, and the environment that converges on the circadian clock to influence susceptibility to developing psychopathology. This model may lend insight into effective treatments for mood disorders and inform development of new interventions.

Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing

The circadian clock in fibroblasts determines the efficiency of wound healing through rhythmic regulation of actin cytoskeletal dynamics. Time heals all wounds Disrupting circadian rhythm, the 24-hour cycle corresponding to light and darkness, is associated with disease and aging. Here, Hoyle et al. discovered a role for circadian control in wound healing. Skin wounds in mice wounded during the circadian rest period healed less quickly than those wounded during the active period. The authors uncovered a circadian regulation of actin, a cytoskeletal protein involved in cell migration, in fibroblasts in the wound-healing response. Analysis of a database of human burn injuries showed that those incurred during the night (rest period) healed more slowly than wounds acquired during the day (active period). This work extends our understanding of cell-autonomous clock control. Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse’s active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.

PKA isoforms coordinate mRNA fate during nutrient starvation

Summary A variety of stress conditions induce mRNA and protein aggregation into mRNA silencing foci, but the signalling pathways mediating these responses are still elusive. Previously we demonstrated that PKA catalytic isoforms Tpk2 and Tpk3 localise with processing and stress bodies in Saccharomyces cerevisiae. Here, we show that Tpk2 and Tpk3 are associated with translation initiation factors Pab1 and Rps3 in exponentially growing cells. Glucose starvation promotes the loss of interaction between Tpk and initiation factors followed by their accumulation into processing bodies. Analysis of mutants of the individual PKA isoform genes has revealed that the TPK3 or TPK2 deletion affects the capacity of the cells to form granules and arrest translation properly in response to glucose starvation or stationary phase. Moreover, we demonstrate that PKA controls Rpg1 and eIF4G1 protein abundance, possibly controlling cap-dependent translation. Taken together, our data suggest that the PKA pathway coordinates multiple stages in the fate of mRNAs in association with nutritional environment and growth status of the cell.