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Modulation of transcription of rRNA genes by rapamycin
1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Abstract:
Lymphosarcoma P1798 cells undergo growth arrest when exponentially growing cultures are exposed to 1 micrograms/ml of Rapamycin (Rapa). This growth arrest is accompanied by inhibition of RNA biosynthesis as measured by incorporation of 3H-uridine into the newly synthesized RNA. Approximately 50% inhibition of 3H-uridine incorporation was observed, upon exposure of P1798 cells to 1 microgram/ml Rapa for 24 h. Run-on transcription experiments using nuclei from Rapa-treated cells indicated a dose-dependent inhibition of transcription or rRNA genes. Cells were relieved from this inhibition of transcription when Rapa was removed from the medium. Under similar conditions, transcriptions of U3 snRNA genes remained unaffected. Cytoplasmic extracts prepared from P1798 cells treated with 1 microgram/ml Rapa for 24 h failed to support transcription from cloned mouse rRNA promoter. This treatment does not affect the RNA polymerase I activity of P1798 cells. Addition of a highly purified murine transcription initiation factor specific for RNA polymerase I reconstitutes the extracts from Rapa-treated P1798 cells. Our data indicate that this new immunosuppressive agent modulates transcription of rRNA genes via regulation of specific transcription factor function.
Insights
Rapamycin (Rapa) inhibits RNA biosynthesis and rRNA gene transcription in lymphosarcoma cells by affecting a specific transcription factor, not RNA polymerase I activity. This effect is reversible upon Rapa removal.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Lymphosarcoma P1798 cells exhibit growth arrest when treated with Rapamycin (Rapa).
- Rapamycin treatment inhibits RNA biosynthesis, evidenced by reduced uridine incorporation.
- The precise mechanism of Rapamycin's effect on RNA synthesis requires elucidation.
Purpose of the Study:
- To investigate the molecular mechanism by which Rapamycin (Rapa) inhibits RNA biosynthesis in lymphosarcoma P1798 cells.
- To determine whether Rapamycin affects transcription initiation or elongation of rRNA genes.
- To identify the specific component within the transcription machinery targeted by Rapamycin.
Main Methods:
- Exposure of exponentially growing P1798 cells to Rapamycin (1 microgram/ml) and measurement of 3H-uridine incorporation.
- Run-on transcription assays using nuclei isolated from Rapamycin-treated cells.
- Analysis of cytoplasmic extracts for rRNA promoter transcription activity and RNA polymerase I activity.
Main Results:
- Rapamycin (Rapa) treatment caused approximately 50% inhibition of 3H-uridine incorporation and rRNA gene transcription.
- Transcription of U3 small nuclear RNA (snRNA) genes remained unaffected by Rapamycin.
- Cytoplasmic extracts from Rapa-treated cells failed to support rRNA promoter transcription, but RNA polymerase I activity was preserved.
Conclusions:
- Rapamycin (Rapa) selectively inhibits the transcription of rRNA genes in lymphosarcoma P1798 cells.
- The drug targets a specific transcription initiation factor for RNA polymerase I, rather than the polymerase itself.
- This study reveals Rapamycin's role in modulating rRNA gene transcription through regulation of transcription factor function.