No abstract

Circadian rhythms are driven by endogenous biological clocks and are synchronized to environmental cues. The chronobiological study of Caenorhabditis elegans, an extensively used animal model for developmental and genetic research, might provide fundamental information about the basis of circadian rhythmicity in eukaryotes, due to its ease of use and manipulations, as well as availability of genetic data and mutant strains. The aim of this study is to fully characterize the circadian rhythm of locomotor activity in C. elegans, as well as a means for genetic screening in this nematode and the identification of circadian mutants. We have developed an infrared method to measure locomotor activity in C. elegans and found that, under constant conditions, although inter-individual variability is present, circadian periodicity shows a population distribution of periods centered at 23.9±0.4 h and is temperature-compensated. Locomotor activity is entrainable by light-dark cycles and by low-amplitude temperature cycles, peaking around the night-day transition and day, respectively. In addition, lin-42(mg152) or lin-42(n1089) mutants (bearing a mutation in the lin-42 gene, homolog to the per gene) exhibit a significantly longer circadian period of 25.2±0.4 h or 25.6±0.5 h, respectively. Our results represent a complete description of the locomotor activity rhythm in C. elegans, with a methodology that allowed us to uncover three of the key features of circadian systems: entrainment, free-running and temperature compensation. In addition, abnormal circadian periods in clock mutants suggest a common molecular machinery responsible for circadian rhythmicity. Our analysis of circadian rhythmicity in C. elegans opens the possibility for further screening for circadian mutations in this species.

Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase.fruit fly | ribosome | dioxygenase | proline | tranlational stress I ron [Fe(II)]-and 2-oxoglutarate (2OG)-dependent oxygenases are a superfamily with diverse biochemical and biological functions. During 2OG oxygenase catalysis, substrate oxidation is coupled to decarboxylation of 2OG, yielding succinate and carbon dioxide (1, 2). Structural studies reveal that the catalytic domain of 2OG oxygenases contains a conserved double-stranded β-helix (DSBH) fold presenting an HXD. . .H facial triad motif that coordinates an Fe(II) cofactor (3, 4). These and other structural features have been used to predict the existence of multiple uncharacterized 2OG oxygenases. In contrast to microorganisms and plants where 2OG oxygenases catalyze a wide variety of oxidative reactions, in animals their biochemical activities appear limited to hydroxylations or demethylations via hydroxylation (1,5,6). Despite progress in making biochemical assignments, the physiological roles of most 2OG oxygenases predicted by bioinformatic analysis of animal genomes are unknown. For instance, we have identified ∼50 putative 2OG oxygenases in the Drosophila genome, but only a few are characterized (7,8).The function of Fatiga, the single Drosophila homolog of human hypoxia inducible transcription factor (HIF) prolyl-4-hydroxylases (PHDs), has been well studied in the context of oxygen sensing (9). HIF prolyl-hydroxylation plays a central role in the animal hypoxic response via hydroxylation of HIF, a posttranslational modification that signals for HIF-α degradation in a physiologically relevant oxygen-dependent manner (10, 11). Given the tractability of these enzymes as targets for pharmacological modulation by 2OG analogs and related co...