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Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture
Abstract:
Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.
Insights
Alpha-amanitin significantly inhibits RNA synthesis in mouse embryos, impacting both total RNA and specific RNA types like polyadenylated and ribosomal RNA. This suggests complex regulatory mechanisms controlling RNA production and processing during early development.
Area of Science:
- Developmental Biology
- Molecular Biology
- Toxicology
Background:
- Early embryonic development relies heavily on precise gene expression and RNA synthesis.
- Understanding the impact of toxins on these fundamental processes is crucial for developmental toxicology.
Purpose of the Study:
- To investigate the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos.
- To determine the dose- and time-dependence of alpha-amanitin's inhibitory effects.
- To elucidate the specific impact on different RNA types and RNA polymerase activities.
Main Methods:
- Exposure of cultured mouse embryos to varying concentrations of alpha-amanitin.
- Measurement of total RNA synthesis via [3H]uridine incorporation.
- Assay of DNA-dependent RNA polymerase activity in embryonic lysates.
- Electrophoretic analysis of RNA synthesis and maturation.
Main Results:
- Alpha-amanitin dose-dependently suppressed total RNA synthesis.
- Polyadenylated RNA synthesis was abolished at concentrations causing minimal total RNA suppression.
- Inhibition of both synthesis and maturation of 45S rRNA precursor was observed, affecting cytoplasmic 28S and 18S RNA.
- Suppression of RNA polymerase II activity was noted, but nucleolar RNA synthesis inhibition was disproportionate to polymerase activity changes.
Conclusions:
- Alpha-amanitin is a potent inhibitor of RNA synthesis in early mouse embryos.
- The drug affects multiple RNA synthesis pathways, including polyadenylated and ribosomal RNA.
- Regulation of nucleolar RNA synthesis and processing may involve factors beyond direct RNA polymerase activity, potentially controlled by nucleoplasmic gene products.