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RESEARCH PRODUCT

Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges

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Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro , change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin protein was found showing a poly(A)-specific 3′-exoribonuclease activity. In addition, the cDNA of the glycogenin gene was identified for subsequent expression studies. Antibodies against nocturnin were raised and used in parallel with the cDNA to determine the regional expression of nocturnin in intact sponge specimens; the highest expression of nocturnin was seen in the epithelial layer around the aquiferous canals. Quantitative PCR analyses revealed that primmorphs after transfer from light to dark show a 10-fold increased expression in the nocturnin gene. In contrast, the expression level of glycogenin decreases in the dark by 3– 4-fold. Exposure of primmorphs to light causes a decrease in nocturnin transcripts and a concurrent increase in glycogenin transcripts. It was concluded that sponges are provided with the molecular circadian clock protein nocturnin that is highly expressed in the dark where it controls the stability of a key metabolic enzyme, glycogenin. Abbreviations: BMAL1, brain and muscle ARNT (aryl hydrocarbon receptor nuclear translocator)-like 1; CLOCK, circadian locomotor output cycles kaput; CRY, cryptochrome; Cy5, indodicarbocyanine; DAPI, 4′,6-diamidino-2-phenylindole; DIG, digoxigenin; GABA, γ-aminobutyric acid; hnRNA, heterogeneous nuclear RNA; ORF, open reading frame; Per, period; qRT–PCR, quantitative real-time reverse transcription PCR; Ror, retinoic acid-related orphan receptor; SDGAPDH, Suberites domuncula glycerol 3-phosphate dehydrogenase; SDGYG, S. domuncula glycogenin; SDNOC, S. domuncula nocturnin