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Regulation of ppp(A2'p)nA-dependent RNase levels during interferon treatment and cell differentiation
Abstract:
The intracellular effector oligonucleotides ppp(A2'p)nA (n = 2- greater than or equal to 4) regulate the breakdown of RNA by activating ppp(A2'p)nA-dependent RNase. Cellular levels of this RNase were demonstrated to be regulated during differentiation of murine embryonal carcinoma cells. An induction of this RNase by interferon was demonstrated in each of three differentiated cell types (F9 clone 9, PYS, and PSA 5E) by analyzing rRNA breakdown following the introduction of ppp(A2'p)nA into the intact cells. In contrast, in three undifferentiated embryonal carcinoma cell lines (F9, PC13 clone 5, and Nulli 2A) there was little if any ppp(A2'p)nA-dependent RNase either with or without interferon pretreatment. These results were confirmed by affinity labeling of the RNase in cell-free systems. Addition of the proteinase inhibitor, leupeptin, to the cell lysis buffer was necessary to stabilize the RNase against cleavage to discrete breakdown products. Moreover, during differentiation of PC13 clone 5 cells by retinoic acid and N6,O2'-dibutyryl-adenosine 3',5'-monophosphate there was a gradual induction of ppp(A2'p)nA-dependent RNase. The expression of this RNase is, therefore, greatly enhanced during cell differentiation. In addition, the double-stranded-RNA-dependent protein kinase was investigated and was found to be interferon-inducible in all of the cell lines regardless of the state of cell differentiation.
Insights
Cellular levels of ppp(A2'p)nA-dependent RNase are regulated during cell differentiation. This RNase is induced by interferon in differentiated cells but not in undifferentiated cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The ppp(A2'p)nA-dependent RNase regulates RNA breakdown.
- Cellular levels of this RNase are modulated during cell differentiation.
- Interferon can influence RNase activity.
Purpose of the Study:
- To investigate the regulation of ppp(A2'p)nA-dependent RNase during cell differentiation.
- To determine the effect of interferon on RNase levels in differentiated and undifferentiated cells.
Main Methods:
- Analysis of rRNA breakdown in intact cells after ppp(A2'p)nA introduction.
- Affinity labeling of RNase in cell-free systems.
- Use of proteinase inhibitor (leupeptin) for RNase stabilization.
Main Results:
- ppp(A2'p)nA-dependent RNase was induced by interferon in differentiated murine embryonal carcinoma cells (F9 clone 9, PYS, PSA 5E).
- Undifferentiated cell lines (F9, PC13 clone 5, Nulli 2A) showed little RNase activity, even after interferon treatment.
- RNase levels gradually increased during differentiation induced by retinoic acid and dibutyryl-cAMP.
- Double-stranded RNA-dependent protein kinase was interferon-inducible in all cell lines, irrespective of differentiation state.
Conclusions:
- Expression of ppp(A2'p)nA-dependent RNase is significantly enhanced during cell differentiation.
- Cell differentiation state plays a crucial role in the regulation of this specific RNase.
- Interferon's effect on this RNase is dependent on the cell's differentiation status.