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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
Published on: February 18, 2022
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Analysis of a Novel Human Protein, ORF3, Encoded by Spacer rDNA
Yaohui Chen1, Jung-Hyun Kim2, Hee-Sheung Lee2
1Laboratory of Genetics and Genomics, Biomedical Research Center, National Institute on Aging, National Institutes of Health Intramural Research Program (NIH IRP), 251 Bayview Blvd., Baltimore, MD, 21224, USA.
Journal of Molecular Evolution
|September 19, 2025
Summary
Researchers identified ORF3, a novel protein encoded by human ribosomal DNA. This protein, found in the cytoplasm and nucleus, interacts with RNA and is upregulated in stressed cells, suggesting a role in cellular responses.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Human ribosomal DNA (rDNA) intergenic spacers contain open reading frames (ORFs).
- One such ORF, named ORF3, encodes a 22 kDa protein.
Purpose of the Study:
- To characterize the novel ORF3 protein encoded by human rDNA.
- To investigate the evolutionary history and cellular localization of ORF3.
- To explore potential functional associations of ORF3 with cellular stress.
Main Methods:
- Isolation of ORF3 clones from mouse:human hybrid cell lines.
- Sequence analysis of ORF3 in human and non-human primate genomes.
- Expression of V5-tagged ORF3 in HEK293 cells and detection using antibodies.
- Cellular localization studies, including RNase A treatment.
- Analysis of ORF3 expression in viral-transformed and senescent cells.
Main Results:
- Full-length ORF3 copies are primarily located on chromosome 21 in human cells, with variations including frameshifts and deletions.
- ORF3 shows high conservation in bonobos but only fragmented homology in other great apes, indicating evolutionary divergence.
- The ORF3 protein localizes to both the cytoplasm and nucleus, with nuclear exclusion upon RNase A treatment suggesting RNA complex formation.
- ORF3 expression is upregulated in viral-transformed HEK293 cells and in human diploid fibroblasts undergoing senescence induced by various stressors.
Conclusions:
- ORF3 is a novel, evolutionarily conserved protein associated with human rDNA.
- Its RNA-binding capability and upregulation under cellular stress suggest a role in cellular response pathways.
- Further research is needed to elucidate the precise function of ORF3.
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