Vascular plants appeared ~410 million years ago then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes (1). We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first non-seed vascular plant genome reported. By comparing gene content in evolutionary diverse taxa, we found that the transition from a gametophyte- to sporophyte-dominated life cycle required far fewer new genes than the transition from a non-seed vascular to a flowering plant, while secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in post-transcriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the tasiRNA pathway and extensive RNA editing of organellar genes.

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Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems.

To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein-protein interactions have become necessary. We describe a novel optical technique for monitoring protein-protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian Pyrearinus termitilluminans). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirational library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with beta-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and beta-arrestin connected with the luciferase fragments, we demonstrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5-10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complementation method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals.

SUMMARYIn most land plants RNA editing frequently occurs in many organelle transcripts, but little is known about the molecular mechanisms of the organelle RNA editing process. In this study, we have characterized the Physcomitrella patens PpPPR_71 gene that is required for RNA editing of the ccmFc transcript. This transcript harbors two RNA editing sites, ccmF-1 and ccmF-2, that are separated by 18 nucleotides. Complementary DNA sequence analysis of ccmFc suggested that RNA editing at the ccmF-1 site occurred before ccmF-2 editing. RNA editing of the ccmF-2 downstream site was specifically impaired by disruption of the PpPPR_71 gene that encodes a polypeptide with 17 pentatricopeptide repeat motifs and a C-terminal DYW domain. The recombinant PpPPR_71 protein expressed in Escherichia coli specifically bound to the 46-nucleotide sequence containing the ccmF-2 editing site. The binding affinity of the recombinant PpPPR_71 was strongest when using the edited RNA at ccmF-1. In addition, the DYW domain also binds to the surrounding ccmF-2 editing site. We conclude that PpPPR_71 is an RNA-binding protein that acts as a site recognition factor in mitochondrial RNA editing.

Dysfunction of circadian clocks exacerbates various diseases, in part likely due to impaired stress resistance. It is unclear how circadian clock system responds toward critical stresses, to evoke life-protective adaptation. We identified a reactive oxygen species (ROS), H2O2 -responsive circadian pathway in mammals. Near-lethal doses of ROS-induced critical oxidative stress (cOS) at the branch point of life and death resets circadian clocks, synergistically evoking protective responses for cell survival. The cOS-triggered clock resetting and pro-survival responses are mediated by transcription factor, central clock-regulatory BMAL1 and heat shock stress-responsive (HSR) HSF1. Casein kinase II (CK2) –mediated phosphorylation regulates dimerization and function of BMAL1 and HSF1 to control the cOS-evoked responses. The core cOS-responsive transcriptome includes CK2-regulated crosstalk between the circadian, HSR, NF-kappa-B-mediated anti-apoptotic, and Nrf2-mediated anti-oxidant pathways. This novel circadian-adaptive signaling system likely plays fundamental protective roles in various ROS-inducible disorders, diseases, and death.

Pentatricopeptide repeat (PPR) proteins are encoded by the nuclear genome as a large gene family in land plants. PPR proteins play essential roles in organelle-related functions, mostly in RNA-processing steps in plastids and mitochondria. In the moss Physcomitrella patens, there is also a large gene family, but the moss PPR proteins are likely to be divergent from those of higher plants. To investigate the function of plastid PPR proteins, we have generated and characterized a PPR protein gene disruptant of P. patens. The PPR531-11-disrupted mosses displayed abnormal phenotypic characteristics, such as a significantly smaller protonemal colony, different chloroplast morphology, and incomplete thylakoid membrane formation. In addition, the quantum yield of photosystem II was reduced in the disrupted mosses. To further investigate whether disruption of the PPR531-11 gene affects chloroplast gene expression, we performed Northern blot and reverse transcription polymerase chain reaction analyses. These analyses revealed that PPR531-11 has a role in intergenic RNA cleavage between clpP and 5-rps12 and in the splicing of clpP pre-mRNA. Western blot analysis showed that disruption of PPR531-11 resulted in a reduced level of ClpP, photosystem II reaction center protein D1, and the stromal enzyme, ribulose-bisphosphate carboxylase/oxygenase. These reductions might result in the severely retarded growth of the protonemal colony. Taken together, we propose a model where PPR531-11 function affects the steadystate level of ClpP, which regulates the formation and maintenance of thylakoid membranes in chloroplasts. This is the first evidence of a PPR protein controlling the protein expression level of ClpP. The pentatricopeptide repeat (PPR)2 is a degenerate 35-amino acid repeating motif that is found in animals, fungi, and plants (1). The PPR motif is similar to but distinct from the tetratricopeptide repeat motif, a well characterized protein interaction motif that is composed of 34 amino acids (2). A particularly large gene family encoding PPR proteins exists in plants, from mosses (3) to flowering plants (4), but not in fungi and animals. For instance, the Arabidopsis thaliana and the rice (Oryza sativa) genomes encode more than 400 PPR proteins. Most plant PPR proteins are predicted to be targeted to the mitochondria or chloroplasts (4). Many PPR proteins play important roles in a wide range of physiological and developmental functions, i.e. cytoplasmic male sterility (5, 6), fertility restoration (7), photosynthesis (8, 9), chloroplast biogenesis (10), and early or late embryogenesis (11,12).Many chloroplast genes of land plants are cotranscribed as polycistronic pre-RNAs, which are then extensively processed into shorter mature RNA species (13). Recently, several lines of evidence that PPR proteins are involved in post-transcriptional regulation in chloroplast gene expression have accumulated. For instance, the maize PPR protein CRP1 is required for intergenic RNA processing of petB and petD dicistronic mRNA (8). The m...

Methods used to assess the efficacy of potentially therapeutic reagents for G protein-coupled receptors (GPCRs) have been developed. Previously, we demonstrated sensitive detection of the interaction of GPCRs and β-arrestin2 (ARRB2) using 96-well microtiter plates and a bioluminescence microscope based on split click beetle luciferase complementation. Herein, using firefly luciferase emitting longer wavelength light, we demonstrate quantitative analysis of the interaction of β2-adrenergic receptor (ADRB2), a kind of GPCR, and ARRB2 in a 96-well plate assay with single-cell imaging. Additionally, we showed bioluminescence in vivo imaging of the ADRB2-ARRB2 interaction in two systems: cell implantation and hydrodynamic tail vein (HTV) methods. Specifically, in the HTV method, the luminescence signal from the liver upon stimulation of an agonist for ADRB2 was obtained in the intact systems of mice. The results demonstrate that this method enables noninvasive screening of the efficacy of chemicals at the specific organ in in vivo testing. This in vivo system can contribute to effective evaluation in pharmacokinetics and pharmacodynamics and expedite the development of new drugs for GPCRs.