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Alternative mechanisms for gene activation induced by poly(rI).poly(rC) and Newcastle disease virus
1Gesellschaft für Biotechnologische Forschung, Abteilung Genetik, Braunschweig, Federal Republic of Germany.
Abstract:
After poly(rI).poly(rC) induction of FS-4 fibroblasts, both human interferon-beta (IFN-beta) mRNA and an additional induced RNA class (12S RNA) hybridize to a genomic cosmid clone containing the human IFN-beta gene as well as 35 kbp of flanking sequences. However, this coinduced 12S RNA does not originate from regions in the neighborhood of the IFN-beta gene, but hybridizes to the genomic cosmid clone via repetitive Alu-family sequences. While IFN-beta mRNA rapidly decays after reaching a maximum 2-4 h after induction, this 12S RNA is stably maintained in the fibroblast cell for more than 16 h. Contrary to IFN-beta mRNA, the level of the 12S RNA is not further elevated by superinduction conditions (cycloheximide treatment) during poly(rI).poly(rC) induction. However, subsequent to treatment with the weaker viral inducer Newcastle disease virus (NDV) both IFN-beta and the 12S RNA transcripts are induced to a higher level in the presence of cycloheximide. Cell-free translation of hybrid-selected 12S RNA leads to detection of an induced protein of 14 kDa. cDNA cloning reveals that the 12S RNA contains part of an Alu-family sequence in the 5'-untranslated region. The 12S RNA is probably not an RNA polymerase III transcript and codes for a protein of 9 kDa (as monitored by in vitro cell-free translation). This discrepancy in molecular mass can be attributed to a retarded migration of the protein in SDS/PAGE.
Insights
Researchers identified a novel 12S RNA in fibroblasts co-induced with human interferon-beta (IFN-beta). This stable 12S RNA, originating from Alu sequences, differs in induction and decay from IFN-beta mRNA and codes for a distinct protein.
Area of Science:
- Molecular Biology
- Virology
- Immunology
Background:
- Poly(rI).poly(rC) is a potent inducer of antiviral responses, including the production of human interferon-beta (IFN-beta).
- Fibroblast cells are a key component of the innate immune system, responding to viral infections and other stimuli.
- Understanding the regulation and function of induced RNAs is crucial for deciphering cellular defense mechanisms.
Purpose of the Study:
- To characterize a co-induced RNA (12S RNA) identified alongside human interferon-beta (IFN-beta) mRNA following poly(rI).poly(rC) induction in FS-4 fibroblasts.
- To investigate the origin, stability, and translational capacity of this novel 12S RNA.
- To compare the regulatory properties of 12S RNA induction with those of IFN-beta mRNA.
Main Methods:
- Hybridization of induced RNA to a genomic cosmid clone containing the IFN-beta gene and flanking sequences.
- Analysis of RNA stability under different induction conditions.
- Cell-free translation of hybrid-selected 12S RNA and cDNA cloning for sequence analysis.
Main Results:
- A novel 12S RNA was co-induced with IFN-beta mRNA but originated from Alu-family repetitive sequences, not the IFN-beta gene neighborhood.
- 12S RNA exhibited distinct stability, persisting for over 16 hours, unlike the rapidly decaying IFN-beta mRNA.
- Cell-free translation and cDNA cloning indicated that 12S RNA codes for a protein of approximately 9-14 kDa, with sequence analysis revealing an Alu-family element in its 5'-untranslated region.
Conclusions:
- The study describes a novel, stable 12S RNA induced in fibroblasts, which is transcriptionally and post-transcriptionally regulated differently from IFN-beta mRNA.
- The 12S RNA's origin from repetitive elements and its distinct expression pattern suggest a potentially unique role in the cellular response to viral inducers.
- The identified protein product of 12S RNA warrants further investigation to elucidate its function in the cellular antiviral state.