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Published on: March 15, 2013
mRNA Decay analysis in Drosophila melanogaster drug-induced changes in glutathione S-transferase D21 mRNA stability
Bünyamin Akgül1, Chen-Pei D Tu
1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Abstract:
We have established an in vivo system to investigate mechanisms by which pentobarbital (PB), a psychoactive drug with a sedative effect, changes the rate of decay of gstD21 mRNA (encoding a Drosophila glutathione S-transferase). Here we describe methods for the use of hsp70 promoter-based transgenes and transgenic lines to determine mRNA half-lives by RNase protection assays in Drosophila. We are able to identify and map putative decay intermediates by cRT-PCR and DNA sequencing of the resulting clones. Our results indicate that the 3'-UTR of gstD21 mRNA is responsive to PB by regulating mRNA decay and that the cis-acting element(s) responsible for the PB-mediated stabilization resides in a 59 nucleotide sequence in the 3'-UTR of the gstD21 mRNA (Akgül and Tu, 2007).
Insights
Pentobarbital (PB) affects gene expression by altering messenger RNA (mRNA) decay rates in Drosophila. A specific DNA sequence in the gstD21 mRNA
Area of Science:
- Molecular Biology
- Genetics
- Pharmacology
Background:
- Pentobarbital (PB) is a psychoactive drug known for its sedative effects.
- Understanding how drugs influence gene expression at the mRNA level is crucial.
- The gstD21 gene encodes a Drosophila glutathione S-transferase, an enzyme involved in detoxification.
Purpose of the Study:
- To establish an in vivo system in Drosophila to investigate the mechanisms of pentobarbital's effect on mRNA decay.
- To determine the half-life of gstD21 mRNA in response to pentobarbital exposure.
- To identify specific DNA sequences responsible for pentobarbital-mediated mRNA stabilization.
Main Methods:
- Utilized hsp70 promoter-based transgenes and transgenic Drosophila lines.
- Quantified mRNA half-lives using RNase protection assays.
- Identified and mapped mRNA decay intermediates via complementary RNA-polymerase chain reaction (cRT-PCR) and DNA sequencing.
Main Results:
- Developed a robust in vivo system for studying mRNA decay in Drosophila.
- Demonstrated that pentobarbital significantly alters the decay rate of gstD21 mRNA.
- Localized the pentobarbital-responsive cis-acting element to a 59-nucleotide sequence within the 3'-untranslated region (3'-UTR) of gstD21 mRNA.
Conclusions:
- The 3'-UTR of gstD21 mRNA plays a critical role in regulating mRNA decay in response to pentobarbital.
- A specific cis-acting element within the 3'-UTR mediates pentobarbital-induced stabilization of gstD21 mRNA.
- This study provides insights into the molecular mechanisms underlying the pharmacogenetic effects of pentobarbital on gene expression.
