Phosphorylation of a Central Clock Transcription Factor Is Required for Thermal but Not Photic Entrainment

Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk(out) flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression

Distribution Analysis of Hydrogenases in Surface Waters of Marine and Freshwater Environments

Background Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism in marine environments is nitrogen fixation. Principal Findings We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H2 uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean. Significance This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H2-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei

The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock

A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins, which is highly dependent on casein kinase Iδ/ε (CKIδ/ε; termed DOUBLETIME [DBT] in Drosophila) and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, β-TrCP (SLIMB in Drosophila). Here we use the Drosophila melanogaster model system, and show that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical SLIMB-binding site on PER. DBT-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located DBT docking site on PER and partially counterbalanced by protein phosphatase activity. We propose that the gradual DBT-mediated phosphorylation of a nonconsensus SLIMB-binding site establishes a temporal threshold for when in a daily cycle the majority of PER proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on PER stability. We also use mass spectrometry to map phosphorylation sites on PER, leading to the identification of a number of “phospho-clusters” that explain several of the classic per mutants

Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function

Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS)

PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients’ behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbt(S) and dbt(L)) in Drosophila