Isolation and characterization of the heat shock RNA 1 - PubMed (original) (raw)
Isolation and characterization of the heat shock RNA 1
Ilya Shamovsky et al. Methods Mol Biol. 2009.
Abstract
The heat shock (HS) response is the major cellular defense mechanism against acute exposure to environmental stresses. The hallmark of the HS response, which is conserved in all eukaryotes, is the rapid and massive induction of expression of a set of cytoprotective genes. Most of the induction occurs at the level of transcription. The master regulator, heat shock transcription factor (HSF, or HSF1 in vertebrates), is responsible for the induction of HS gene transcription in response to elevated temperature. Under normal conditions HSF is present in the cell as an inactive monomer. During HS, HSF trimerizes and binds to a consensus sequence in the promoter of HS genes, stimulating their transcription by up to 200-fold. We have shown that a large, noncoding RNA, HSR1, and the translation elongation factor eEF1A form a complex with HSF during HS and are required for its activation.
Figures
Figure 1
Isolation of HSF1 activating fraction and HSR1 from a lysate of heat shocked HeLa (middle panel) and BHK (left panel) cells. Lane 1 (BHK and HeLa): lysate of heat shocked cells; lane 2 (BHK): supernatant after incubation of the lysate with HSF1-Sepharose beads; lane 3 (BHK) and lane 2 (HeLa): HSF1-Sepharose beads after incubation with a lysate of heat shocked cells and washes; lane 4 (BHK) and lane 3 (HeLa): HSF1-Sepharose beads after three successive elutions at 43°C; lanes 5–7 (BHK) and 4–6 (HeLa): three successive elutions with buffer at 43°C. Right panel: silver stained PAAG of RNA isolated from fractions 5–7 shown in left panel (BHK cells). Where indicated, RNA was treated with DNase I or RNase A prior to loading the gel.
Figure 2
In vitro reconstituted system for the activation of HSF1. Upper panel: recombinant mouse HSF1 (10 nM final) and eEF-1A (100 nM final, omitted as shown) were incubated in the absence of RNA, in the presence of HSR1 isolated from either BHK or HeLa cells, as indicated, in the presence of (25 nM final) in vitro transcribed sense (T3) or antisense (T7) mammalian HSR. Reactions were then incubated with the radiolabeled HSE oligo and separated on a native PAAG, as described in the text. The gel was dried and radioautographed. Lower panel: HSR1 antisense oligos screening. EMSA reactions were assembled as described above with the exception of the inclusion of antisense oligos at a 10-fold molar excess over HSR1.
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