In vertebrates, COX-1 and COX-2, two cyclooxygenase isozymes with different physiological functions and gene regulation, catalyze identical reactions in prostaglandin synthesis. It is still not understood why there are multiple forms of COX enzyme in the same cell type and when the evolutionary duplication of the COX gene occurred. Here we report the structure of two genes encoding for COX isozymes in the coral Gersemia fruticosa, the first non-vertebrate organism from which a cyclooxygenase was characterized. Both genes are about 20 kb in size and consist of nine exons. Intron/exon boundaries are well conserved between coral and mammalian COX genes. mRNAs of the previously reported G. fruticosa COX-A (GenBank™ accession number AY004222) and the novel COX-B share 94% sequence identity in the coding regions and less than 30% in the 5′- and 3′-untranslated region. Transcripts of both COX genes are detectable in coral cells, although the transcriptional level of COX-A is 2 orders of magnitude higher than COX-B. Expression of both coral genes in mammalian cells gave functional proteins with similar catalytic properties. By data base analyses we also detected and constructed different pairs of COX genes from the primitive chordates, Ciona savignyi and Ciona intestinalis. These two gene pairs encode proteins with 50% intra-species and only 70% cross-species sequence identity. Our results suggest that invertebrate COX gene pairs do not correspond to vertebrate COX-1 and COX-2 and are consistent with duplication of the COX gene having occurred independently in corals, ascidians, and vertebrates. It is evident that due to the importance and complexity of its regulatory role, COX has multiple isoforms in all organisms known to express it, and the genes encoding for the isozymes may to be regulated differently.

In vertebrates, the synthesis of prostaglandin hormones is catalyzed by cyclooxygenase (COX)-1, a constitutively expressed enzyme with physiological functions, and COX-2, induced in inflammation and cancer. Prostaglandins have been detected in high concentrations in certain corals, and previous evidence suggested their biosynthesis through a lipoxygenase-allene oxide pathway. Here we describe the discovery of an ancestor of cyclooxygenases that is responsible for prostaglandin biosynthesis in coral. Using a homology-based polymerase chain reaction cloning strategy, the cDNA encoding a polypeptide with ∼50% amino acid identity to both mammalian COX-1 and COX-2 was cloned and sequenced from the Arctic soft coral Gersemia fruticosa. Nearly all the amino acids essential for substrate binding and catalysis as determined in the mammalian enzymes are represented in coral COX: the arachidonate-binding Arg¹²⁰ and Tyr³⁵⁵ are present, as are the heme-coordinating His²⁰⁷ and His³⁸⁸; the catalytic Tyr³⁸⁵; and the target of aspirin attack, Ser⁵³⁰. A key amino acid that determines the sensitivity to selective COX-2 inhibitors (Ile⁵²³ in COX-1 and Val⁵²³ in COX-2) is present in coral COX as isoleucine. The conserved Glu⁵²⁴, implicated in the binding of certain COX inhibitors, is represented as alanine. Expression of the G. fruticosa cDNA afforded a functional cyclooxygenase that converted exogenous arachidonic acid to prostaglandins. The biosynthesis was inhibited by indomethacin, whereas the selective COX-2 inhibitor nimesulide was ineffective. We conclude that the cyclooxygenase occurs widely in the animal kingdom and that vertebrate COX-1 and COX-2 are evolutionary derivatives of the invertebrate precursor.

The human WBSCR22 protein was previously shown to be up-regulated in invasive breast cancer and its ectopic expression enhances tumor cell survival in the vasculature. In the current study, we show that the WBSCR22 protein is important for cell growth. Knock-down of WBSCR22 with siRNA results in slower growth of WBSCR22-depleted cells. Treatment with siWBSCR22 causes defects in the processing of pre-rRNAs and reduces the level of free 40S ribosomal subunit, suggesting that WBSCR22 is involved in ribosome small subunit biosynthesis. The human WBSCR22 partially complements the growth of WBSCR22 yeast homologue, bud23 deletion mutant suggesting that the human WBSCR22 is a functional homologue of yeast Bud23. WBSCR22 is localized throughout the cell nucleus and is not stably associated with ribosomal subunits within the cell nucleus. We also show that the WBSCR22 protein level is decreased in lymphoblastoid cell lines derived from William-Beuren Syndrome (WBS) patients compared to healthy controls. Our data suggest that the WBSCR22 protein is a ribosome biogenesis factor involved in the biosynthesis of 40S ribosomal particles in mammalian cells.

The human WBSCR22 protein is a 18S rRNA methyltransferase involved in pre-rRNA processing and ribosome 40S subunit biogenesis. Recent studies have shown that the protein function in ribosome synthesis is independent of its enzymatic activity. In this work, we have studied the WBSCR22 protein interaction partners by SILAC-coupled co-immunoprecipitation assay and identified TRMT112 as the interaction partner of WBSCR22. Knock-down of TRMT112 expression decreased the WBSCR22 protein level in mammalian cells, suggesting that the stability of WBSCR22 is regulated through the interaction with TRMT112. The localization of the TRMT112 protein is determined by WBSCR22, and the WBSCR22-TRMT112 complex is localized in the cell nucleus. We provide evidence that the interaction between WBSCR22/Bud23 and TRMT112/Trm112 is conserved between mammals and yeast, suggesting that the function of TRMT112 as a co-activator of methyltransferases is evolutionarily conserved. Finally, we show that the transiently expressed WBSCR22 protein is ubiquitinated and degraded through the proteasome pathway, revealing the tight control of the WBSCR22 protein level in the cells.