Ribosomal oxygenase 1 is a protein that in humans is encoded by the RIOX1 gene (previously NO66). The protein is involved in ribosome biogenesis and replication and regulation of heterochromatic regions of the nucleus[5]. It is a chromatin regulator, and a regulator of osteoblast differentiation[6]. It is a Jumonji C (JmjC) domain-containing protein[7] in the subgroup of ribosomal oxygenases (ROXs), part of the superfamily of Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases[8]. Ribosomal oxygenase 1 It is a dual-location protein found in the nucleus, nucleolus, and nucleoplasm of the cell.[9] RIOX1 is a single-exon-gene, intronless, and is found across several species. The gene is involved in gene transcription regulation in the nucleus of the cell.[9]Expression of RIOX1 correlates with potential of cancer. Expression levels are shown to closely correlate with the malignant potential of renal cell tumors and colorectal cancer. The gene has been shown to be an oncogenic driver (initiates cancer cell growth and proliferation) of prostate cancer.[10]
Human RIOX1 exon structureDomain architecture for RIOX1 and RIOX2
Both RIOX1 and RIOX2 (Previously MINA53) modify ribosome proteins through histidine hydroxylation,[8] specifically His 216.[7] The two proteins differ in the presence of the N-terminal extension which is absent in RIOX2.[8] They are both part of the JmjC-domain-containing protein family, which are a part of a class of histone demethylases, which control the structure of chromatin in order to regulate gene expression.[10] JmjC histone demethylases are linked to human diseases, many cases in cancer and neurological disorders. It enables protein demethylase and peptidyl-histidine dioxygenase activity. It also is involved in regulating DNA repair.[11] RIOX1 has a unique N-terminal extension domain, meaning it can have unique interactions with other molecules, in addition to assistance in protein folding. Nuclear distribution of RIOX1 is localized to perichromosomal (specialized chromosome domain containing proteins for certain processes[12]) cytoplasm in metaphase and early anaphase, localized to chromosomes in late anaphase, and localized to prenucleolar bodies in late telophase, during cellular processes of MCF-7 cells.[5]
RIOX1/NO66 hydroxylates histidine residues in the ribosomal protein RPL8Ramachandran Plot for the RIOX1 protein, Crystal Structure of human NO66
Phylogenetics
Analysis of RIOX1 and RIOX2 orthologous genes from different species show presence of the gene in early Cnidaria. The genomic sequence exhibited presence of both RIOX1 and RIOX2 in Hydra vugaris, an early metazoan in the phylum Cnidaria.[8] In addition to presence in Cnidaria, RIOX1 was found in the genes of Arthropoda, Annelida, Nematoda and Mollusca, where RIOX2 was not found, with the exception of Priapulida. RIOX1's presence across species suggests that the gene is phylogenetically older, from which RIOX2 was evolved. RIOX2 is thought to have evolved in Chordata.
Structure
RIOX1 has a length of 2429 base pairs. It consists of 641 amino acids[13].Structure of the protein shows active sites within the JmjC domains, a dimerization domain, and a winged-helix domain.[8] The crystal structure of RIOX1 folds into a functional tetrameric formation.[14] Research on the structure of NO66 [9]showed the JmjC domain from residues 176-426, hinge-domain from residues 427-510, beta hairpin motif from residues 511-547, and the C-terminal wHTH motif from residues 548-510. The dimer interface consists of two similar molecules in one asymmetrical unit (main-chain r.m.s.d. = 0.22Å).[9]Both the JmjC and C-terminal domains are necessary in tetrameric assembly (compilation of four individual subunits or monomers).[14] The JmjC domain can form stable dimers when interacting with C-terminal helices. RIOX1 contains a region with similarities with the COG2850 (cluster of orthologous groups of proteins).[5]
RIOX1 and RIOX2 differ in the presence of the N-terminal extension, which is not found in RIOX2. The N-terminal extension is predicted to have a nuclear localization signal, which is an amino acid sequence that can tag proteins for transport into the cell's nucleus.[8]
The JmjC domain found in the RIOX1 gene is a part of the regulatory network of Osterix. RIOX1 interacts with Osterix and inhibits Osterix-dependent promoter activities.[14] Osterix is an osteoblast-specific transcription factor necessary for differentiation in osteoblasts and bone formation. The oligomerization (monomers combining) interface of RIOX1 interacts with a fragment of Osterix. The amino acid residues that form the Osterix binding groove are highly conserved in different vertebrate species, which indicates that RIOX1 plays a role in regulation of the differentiation of osteoblasts and bone formation by interaction with Osterix.[14] Hinge domain-dependent oligomerization of RIOX1 is also necessary in inhibition of Osterix dependent gene activation.[14]
Location
Ribosomal oxygenase 1 It is a dual-location protein found in the nucleus, nucleolus, and nucleoplasm of the cell.[9] The protein has a large accumulation in nuclei as well as in intranuclear bodies of late replicating chromatin[5]. Ribosomal oxygenase 1 is localized to perichromosomal cytoplasm in metaphase and early anaphase. It is localized to chromosomes in late anaphase and localized to prenucleolar bodies in late telophase[5].
Cancer
The expression of RIOX1 is linked to cancer potential. It is found to be over-expressed in lung carcinomas[6]. Expression of the gene is shown to correlate with increases in the levels of malignant potential of renal cell tumors and colorectal cancer. Expression of the gene has also been shown to initiates prostate cancer cell growth[10].
Researchers at the German Cancer Research Center sequenced Xenopus laevis oocytes to examine this gene, with the results showing association with large preribosomal particles (>60S), suggesting that the gene plays a role in the assembly and processing of ribosomal subunits.[5]
Investigation into the RIOX1 gene at The University of Texas MD Anderson Cancer Center on mouse embryos shows evidence that over-expression of the gene is associated with osteoporosis in adult mice.[15] Ablation of the RIOX1 gene in cells of the Prx1-expressing mesenchymal (related to embryonic connective tissue) lineage causes an acceleration of osteochondrogenic (bone or cartilage forming) differentiation and a larger skeleton in adult mice, whereas mesenchyme-specific over-expression of RIOX1 inhibits formation of bone and cartilage resulting in dwarfism and osteopenia. RIOX1 is a chromatin modifier and negatively regulates mesenchymal progenitor differentiation, as well as Osterix activity.[15]
Research into oncogenic and osteolytic functions of RIOX1 in castration-resistant prostate cancer showed upregulated levels of the gene in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of RIOX1 promoted cell survival and invasion of prostate cancer cells. In contrast, knockdown of RIOX1 resulted in decreased cell survival and increased sensitivity to the chemotherapy medication docetaxel.[16]
Other Research
Analysis of healthy human hematopoietic cells compared to Acute Myeloid Leukemia cell lines found that expression levels of RIOX1 closely correlates with the malignant potential of colorectal cancer and renal cell tumors.[10] Acute Myeloid Leukemia (AML) is caused by the malignant transformation of hematopoietic (blood forming) stem cells, in cases of loss of differentiation and uncontrolled proliferation.[10] The RIOX1 gene was not present in the AML KG1/ KG1a cell lines, and researchers found that the lack of the gene in these cells is due to hyper-methylation of the promoter. This is referred to as a "stem cell-like" system.[10] Ectopic (unusual) expression of RIOX1 in KG1a cells inhibits proliferation and rDNA transcription. The gene impacts the transition from stem cells to progenitors (differentiated cells) by modification or regulation of gene expression.[10]
There is evidence that the loss of the RIOX1 restores cell proliferation and reduces cell death after exposure to ionizing radiation. Radiation causes damage to DNA, and influence of epigenetic modifications affect the efficiency of repair to DNA. DNA damage is cytotoxic and becomes lethal when not repaired[11].Double strand breaks are harmful, and can be repaired through homologous recombination or nonhomologous end-joining. Removal of the RIOX1 gene improves levels of homologous recombination repair but not nonhomologous end-joining repair in irradiated oral mucosal epithelial cells and irradiated bone marrow cells.[11] High expression of RIOX1 maintains methylation of the cyclic GMP-AMP synthase (cGAS) protein at lysine residue 491 at a low level, and therefore impedes homologous recombination repair and decreases cellular tolerance to ionizing radiation. A RIOX1 dependent mechanism involved in the function of this cGAS enzyme is necessary for the regulation of ionizing radiation-elicited homologous recombination repair.[11] RIOX1 was also shown to abolish the monomethylation of K491 in cGAS, disrupting interactions between cGAS and SGF29. Without the K491 monomethylation, cGAS binds to poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase 1 and blocks homologous recombination repair.[11]
Research at the Shanghai Synchrotron Radiation Facility purified the RIOX1 protein in order to determine the structure, perform GST pull-down assay, hydroxylation activity analysis, size-exclusion chromatography, and isothermal titration calorimetry. Results indicated that oligomerization is essential in the function if catalytic activity in the protein.[9] Results also indicated that tetramerization is required for the protein to catalyze hydroxylation effectively.
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