Analysis of a Novel Human Protein, ORF3, Encoded by Spacer rDNA

An open reading frame in the intergenic spacer of human ribosomal (r)DNA codes for a 190 amino acid, 22 kDa protein that we have named ORF3. It comprises a 5'AluSx repeat sequence encoding 96 amino acids followed by a stretch of 94 amino acids containing a unique repeated stretch of 5 hydrophobic residues. Full copies of ORF3 have been isolated as transformation-associated recombination clones from mouse:human hybrid cell lines containing human chromosomes 21 or 22. In initial instances where the chromosome complement of rDNA repeats is fully resolved in whole genomes, in CHM13 cells, complete copies of ORF3 are mainly concentrated in a tandem cluster on chromosome 21, while other chromosomes contain 1 or 2 full copies, with the sequence in other rDNA repeats interrupted by a frameshift mutation. A diploid cell complement (HG002) again has both complete open reading frames (ORFs) and other copies with the frameshift or deletions. In searches among non-human primate sequences to assess the evolutionary history of ORF3, a > 93% conserved copy of the full sequence of the ORF, as well as copies with in-frame deletions, was found in bonobo, but only fragments homologous to the ORF were seen in chimpanzee, orangutan, and gorilla rDNA examined thus far. ORF3 was expressed as a V5-tagged chimeric protein in human kidney epithelial HEK293 cells, and both ORF3-V5 and endogenous ORF3 were detected with a newly generated antibody. The protein is found in both cytoplasm and nucleus. However, upon treatment of cells with RNase A, the protein is excluded from the nucleus, suggesting that it is in complexes with RNA. Although any function is currently unknown, the ORF3 protein is upregulated, speculatively associated with changes in chromatin, in viral-transformed HEK293 cells and in human diploid fibroblast cells rendered senescent by treatment with etoposide, ionizing radiation, or an oxidant (H₂O₂).