Regulation of mRNA stability through a pentobarbital-responsive element

Abstract

Pentobarbital, a general anesthetic and non-genotoxic carcinogen, can induce gene expression by activating transcription. In the Drosophila glutathione S-transferase D21 (gstD21) gene, pentobarbital's regulatory inXuence extends to the level of mRNA turnover. Transcribed from an intronless gene, gstD21 mRNA is intrinsically very labile. But exposure to pentobarbital renders it stabilized beyond what can be attributed to transcriptional activation. We aim here to identify cis-acting element(s) of gstD21 mRNA as contributors to the molecule's pentobarbital-mediated stabilization. In the context of hsp70 5ЈUTR and the 3ЈUTR of act5C, gstD21 mRNA, minus its native UTRs, is stable. Maintaining the same context of heterologous UTRs, we can reconstitute using the full-length gstD21 sequence the inherent instability of gstD21 mRNA and its stabilization by pentobarbital. Transgenic Xies that express these chimeric gstD21 mRNA exhibit decay intermediates lacking 3ЈUTR, which are not stabilized by PB treatment. The 3ЈUTR sequence, when inserted downstream from a reporter transcript, stabilizes it 1.6-fold under PB treatment. The analysis of the decay intermediates suggests a polysomeassociated decay pattern. We propose a regulatory model that features a 59-nucleotide pentobarbital-responsive element (PBRE) in the 3ЈUTR of gstD21 mRNA.

Chapter 15 mRNA Decay Analysis in Drosophila melanogaster

“…This method is based on circularization of decapped mRNAs followed by RT-PCR (Couttet et al, 1997) (Also see Chapter 22 by Grange). Although several methods are available to identify the 5 0 -or 3 0 -end of an mRNA individually, we have successfully used circular RT-PCR to simultaneously map both ends of various gstD21s at the nucleotide level (Akgül and Tu, 2007). It has been particularly useful in mapping multiple gstD21 decay intermediates at the nucleotide level.…”

“…It has been particularly useful in mapping multiple gstD21 decay intermediates at the nucleotide level. We first use a nested set of radioactive probes in RPA analyses to roughly estimate the ends of the intermediates (Akgül and Tu, 2007). These results are then used as a guide in designing the appropriate pair of primers ($50 nt from the ends) for the subsequent RT-PCR experiment.…”

“…Cloned plasmid DNAs are randomly selected for sequence analysis to determine the 5 0 -and/or 3 0 -ends of the mRNAs. We sequenced $100 clones to obtain a distribution of the ends of RNA decay intermediates of gstD21 mRNA (Akgül and Tu, 2007).…”

“…In the following, we describe a system to investigate mRNA decay under the effects of PB using the gstD21 mRNA as the reporter. Furthermore, by incorporating the chimeric gene approach, we are able to dissect the D21 mRNA for the cis-acting elements responsible for drugmediated stabilization of gstD21 mRNAs in Drosophila (Akgül and Tu, 2007).…”

Regulatory element identification in subsets of transcripts: Comparison and integration of current computational methods

“…Post-transcriptional control is involved in regulation of many physiological and pathological processes, including normal development, homeostatic functions, and carcinogenesis. Information embedded in the messenger ribonucleic acid (mRNA) sequence is used by the cell to regulate groups of transcripts (designated post-transcriptional operons or regulons) (Keene 2007) and has been mechanistically characterized in transcription termination (Brambilla et al 1997), mRNA localization (Macdonald et al 1993;Kloc et al 2000;Chabanon et al 2004), stability (Brewer 1991;Theil 1993;Akgul and Tu 2007;Vlasova et al 2008), alternative splicing (Massiello et al 2004), and ribosome recruitment (Larsson et al 2006).…”