HSF1-TPR Interaction Facilitates Export of Stress-induced HSP70 mRNA

Hollie S Skaggs,Hongyan Xing,Donald C Wilkerson,Lynea A Murphy,Yiling Hong,Christopher N Mayhew,Kevin D Sarge

doi:10.1074/jbc.m704054200

Abstract

Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear pore-associating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.

Highlights

HSF1 is the transcription factor responsible for up-regulating transcription of HSP70 and other genes in response to elevated temperature and other stress conditions
The results indicate that cells transfected with GFP-TPR-(14 –117) or GFPTPR-(1218 –1320) exhibited a decrease in the cytoplasmic levels of luciferase mRNA expressed from the heat shock elementcontaining HSP70 promoter compared with cells transfected with GFP (Fig. 5C, upper panels)
The results presented in this work indicate that, in response to stress, the TPR protein interacts with the stress gene transcriptional regulator HSF1, is recruited to the HSP70 promoter region, and preferentially associates with mRNAs transcribed from this promoter compared with those expressed from a non-stress-induced promoter and that the HSF1-TPR interaction is required for efficient export of HSP mRNAs from the nucleus during stress

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Assay—The interactions between pGBDHSF1 and pVP16-TPR-(14 –117) and pVP16-TPR-(1218 – 1320) were characterized by streaking yeast (strain pJ694A) containing these constructs or pGBD-HSF1 bait and empty pVP16 plasmid (as a negative control) on plates lacking tryptophan and leucine; tryptophan, leucine, and histidine; or tryptophan, leucine, and alanine. The template for in vitro transcription was created by PCR with the HSP70-luciferase plasmid and primers 5Ј-cacggaaagacgatgacg-3Ј and 5Ј-taatacgactcactataggttgggtaacgccaggg-3Ј This PCR product contained the 3Ј-end of the luciferase mRNA, ending with the polyadenylation signal (yielding a protected fragment of 325 bp) and untranscribed vector sequence (resulting in an unprotected fragment of 438 bb). Cells were resuspended in 2 ml of low stringency radioimmune precipitation assay (RIPA) buffer (50 mM TrisHCl (pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, 1ϫ Complete protease inhibitor mixture (Roche Applied Science), and 80 units of RNaseOUT (Invitrogen)), pipetted 20 times, and incubated on ice for 10 min. ((Ct(IgG)ϪCt(Input))Ϫ(Ct(TPR)ϪCt(Input))) For the experiments analyzing luciferase cDNA, primers used for quantitative PCR analysis were 5Ј-gtctgaattccagtcgatgtacacgttcg-3Ј and 5Ј-cacgaagcttgcatgcgagaactccacgc-3Ј. The results are graphed as relative differences between cytoplasmic and nuclear HSP70 mRNA levels compared with the control samples (GFP alone), which were set to a value of 1

RESULTS

Relative Tpr association

DISCUSSION