NSF Award
9507328
MCB-9507318 Brodl To study the cell biology of higher plants' responses to environmental stress, a model system for investigation is the heat shock response of barley aleurone cells. Normally aleurone cells are dedicated to protein secretion; however, heat shock dramatically redirects their cellular activities. Heat shock abruptly arrests secretory protein synthesis, yet nonsecretory protein synthesis continues. This is accomplished by the selective destabilization of otherwise stable secretory protein mRNAs. Heat shock also causes the loss of the stacked cistemal ER lamellae upon which secretory protein mRNAs are translated; only short, single tubular ER remain. Ribosome density decreases by 50%. This may provide the discriminatory mechanism or selectively stopping the synthesis of secretory proteins; nonseeretory protein mRNAs are translated by "free" ribosomes. The principal distinction between secretory and nonsecretory protein mRNAs is a region encoding an amino terminal signal sequence that directs secretory protein mRNA translation to take place at the ER surface. This proposal investigates the mechanisms that operate during heat shock to selectively destabilize secretory protein mRNA. There are two central objectives in this work: 1) To determine whether the signal sequence is an important factor in selectively targeting secretory protein mRNA for destabilization. Using PCR the coding region for the signal sequence (ss) of the secretory protein a-amylase will be fused in-frame to the coding region of b-glucuronidase (GUS). Constructs will also be made that include either or both the 5' and 3' untranslated regions (UTRs) of a-amyase such that they flank GUS or ss-GUS. Transcription will be driven by the tetracycline- regulated Top l0 promoter. The constructs will be microprojectile bombarded into intact aleurone layers. The levels of the GUS reporter transcript will be monitored by GUS selected PCR amplification at set intervals following tetrac ycline-induced transcription arrest. 2) To understand something of the fate of secretory protein mRNAs upon heat shock. For this objective two types of experiments will be performed: a) The levels of secretory protein mRNAs in free and ER-bound polysomes before and at selected intervals during heat shock will be monitored in order to learn whether secretory protein mRNAs remain attached to the ER during heat shock. b) UV cross-linking experiments with radiolabeled a-amylase mRNA (including the UTRs) and cell lysates will be performed to investigate whether heat shock induces the binding of RNA binding proteins that may play a role in the destabilization of secretory protein mRNAs. %%% Plants respond to higher-than-normal, non-lethal temperatures by synthesizing heat shock proteins and by transiently shutting down the synthesis of other proteins necessary for normal growth and development. My laboratory works to understand how the shut down is controlled. Our model system for study is aleurone, an outer layer of cells surrounding the cereal grain whose function is to start the breakdown of starch reserves during germination. If the aleurone layer is briefly exposed to higher-than-normal temperature (heat shock), the synthesis of proteins involved this breakdown, including the starch-digesting enzyme, amylase, is shut down. The synthesis of other proteins, those not released (secreted) from the aleurone, continues unabated. Early experiments have determined the cause of this selective cessation of protein synthesis. Messenger RNAs (mRNAs) (transcripts of genetic information encoded by DNA) encoding the secreted proteins are destabilized, but nonsecretory protein mRNAs remain stable. To determine why, focus is brought to bear on another difference between mRNAs for secretory and non-secretory proteins. Secretory proteins frequently have additional amino acids tacked onto one end which are cleaved from the mature protein, but function to target the prote in to the secretory system and are encoded by their mRNA. This sequence of amino acids is called the "signal sequence". Does the signal sequence contribute to the instability of mRNAs encoding secreted proteins? To answer that question, genetic engineering will be used to fuse the signal sequence coded by amylase mRNA to the mRNA encoding an easily assayed (reporter) protein (b-glucuronidase) that is not normally secreted. The stability of the reporter protein mRNA with and without the signal sequence will be monitored during heat shock. If the hypothesis that the signal sequence confers sensitivity to heat is correct, then the proteins which cause destabilization by binding to the signal sequence-containing mRNA during heat shock will be identified. These experiments to deepen our understanding of the plant cellular and genetic response to environmental extremes are of importance to agriculture in both temperate and tropical climates. They are also important because they reveal part of the basic mechanism of cellular adaptation to changes in temperature. ***
