In response to elevated temperatures, cells from many organisms rapidly transcribe a number of mRNAs. In Saccharomyces cerevisiae, this protective response involves two regulatory systems: the heat shock transcription factor (Hsf1) and the Msn2 and Msn4 (Msn2/4) transcription factors. Both systems modulate the induction of specific heat shock genes. However, the contribution of Hsf1, independent of Msn2/4, is only beginning to emerge. To address this question, we constructed an msn2/4 double mutant and used microarrays to elucidate the genomewide expression program of Hsf1. The data showed that 7.6% of the genome was heat-induced. The up-regulated genes belong to a wide range of functional categories, with a significant increase in the chaperone and metabolism genes. We then focused on the contribution of the activation domains of Hsf1 to the expression profile and extended our analysis to include msn2/4⌬ strains deleted for the N-terminal or C-terminal activation domain of Hsf1. Cluster analysis of the heat-induced genes revealed activation domain-specific patterns of expression, with each cluster also showing distinct preferences for functional categories. Computational analysis of the promoters of the induced genes affected by the loss of an activation domain showed a distinct preference for positioning and topology of the Hsf1 binding site. This study provides insight into the important role that both activation domains play for the Hsf1 regulatory system to rapidly and effectively transcribe its regulon in response to heat shock.The ability to respond to a large number of environmental stresses is crucial for the survival of all organisms (1). Heat shock is among a highly diverse group of environmental conditions that alter gene expression in prokaryotic and eukaryotic cells. The response to heat shock is characterized by a rapid induction of a conserved group of heat shock proteins (HSPs).2 In Saccharomyces cerevisiae, two regulatory systems are involved in this response: the heat shock transcription factor (Hsf1) (2, 3) and the Msn2 and Msn4 (Msn2/4) transcription factors (4, 5).Yeast Hsf1 is an essential protein that binds to inverted repeats of nGAAn called heat shock elements (HSEs) within the promoters of many HSPs and activates their transcription. Hsf1 is composed of several well defined domains that are important for its function. They include the highly conserved central core, which is made up of the winged-helix-turn-helix DNA-binding domain (6, 7) and the hydrophobic coiled-coil region essential for the regulation of homotrimer formation (8 -10). In addition, yeast Hsf1 has two trans-activation domains, one at the N terminus and the other at the C terminus (11, 12).Although the structure and function of HSF is generally conserved from various organisms, there is variability in the number and importance of HSF genes in any particular organism. The yeasts, S. cerevisiae and Schizosaccharomyces pombe, have one HSF gene that is essential for cell survival (2, 13, 14), whereas Drosophila melanogaster...
RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the aCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to aCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for aCP decoy action. Because aCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of aCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.
The poly(C)-binding proteins, ␣CPs, comprise a set of highly conserved KH-domain factors that participate in mRNA stabilization and translational controls in developmental and viral systems. Two prominent models of ␣CP function link these controls to late stages of erythroid differentiation: translational silencing of 15-lipoxygenase (Lox) mRNA and stabilization of ␣-globin mRNA. These two controls are mediated via association of ␣CPs with structurally related C-rich 3-untranslated region elements: the differentiation control elements (DICE) in Lox mRNA and the pyrimidine-rich motifs in ␣-globin mRNA. In the present report a set of mRNA translation and stability assays are used to determine how these two ␣CP-containing complexes, related in structure and position, mediate distinct posttranscriptional controls. While the previously reported translational silencing by the DICE is not evident in our studies, we find that the two determinants mediate similar levels of mRNA stabilization in erythroid cells. In both cases this stabilization is sensitive to interference by a nuclear-restricted ␣CP decoy but not by the same decoy restricted to the cytoplasm. These data support a general role for ␣CPs in stabilizing a subset of erythroid mRNAs. The findings also suggest that initial binding of ␣CP to target mRNAs occurs in the nucleus. Assembly of stabilizing mRNP complexes in the nucleus prior to export may maximize their impact on cytoplasmic events.
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