Nuclear Retention Prevents Premature Cytoplasmic Appearance of mRNA Thomas Beuchert Kallehauge, Marie-Cécile Robert, Edouard Bertrand, Torben Heick Jensen  Molecular Cell  Volume 48, Issue 1, Pages 145-152 (October 2012) DOI: 10.1016/j.molcel.2012.07.022 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 HSP104 Dot RNA Release and Translation Profiles (A) Outline of transcriptional pulse-chase experiment. Arrow marks the time of thiolutin addition in (D). (B) High-resolution HSP104 RNA-FISH of WT and mex67-5 samples collected and fixed at the time points indicated in (A). Ten Cy3-labeled DNA oligonucleotide probes directed towards the first 700 nt of HSP104 (top) allowed for single RNA molecule detection. Images were acquired with a 10 ms exposure time for dot signal identification (left panels for each of the two strains) and a 110 ms exposure time for cytoplasmic single molecule detection (middle panels). Longer exposed images were merged with DAPI signal obtained from the same cells (right panels). The fractions of dot-positive mex67-5 cells in the displayed experiment are shown to the right. Dashed lines, within long exposure panels, indicate the cytoplasmic quantification boundary. (C) Quantification of the number of cytoplasmic HSP104 RNA molecules over the experimental time course in WT and mex67-5 cells. Error bars of the individual experiment (number of cells counted in displayed experiment: nwt, 25°C = 226, nwt, 37°C, 10′ = 217, nwt, 10′ at 25°C = 314, nwt, 60′ at 25°C = 364, nwt, 120′ at 25°C = 265; nmex67-5, 25°C = 226, nmex67-5, 37°C, 10′ = 217, nmex67-5, 10′ at 25°C = 314, nmex67-5, 60′ at 25°C = 364, nmex67-5, 120′ at 25°C = 265) represent standard deviations, which are relatively high due to natural bursting of heat shock genes (Blake et al., 2006; Chubb et al., 2006). T-test between mex67-5 37°C and time course: pmex67-5, 37°C, 10′ versus mex67-5, 10′ at 25°C = 9.22E-24, pmex67-5, 37°C, 10′ versus mex67-5, 60′ at 25°C = 6.31E-45, pmex67-5, 37°C, 10′ versus mex67-5, 120′ at 25°C = 8.31E-35. Two biological repeats were performed with a similar outcome. (D) Quantification of cytoplasmic HSP104 RNA in mex67-5 cells treated with 50 μg/mL of the transcriptional inhibitor thiolutin upon return of the cells to 25°C. Standard deviations of individual experiments were calculated as in Figure 1C. Displayed experiment n = 200 cells. T test between mex67-5 37°C and thiolutin time course: pmex67-5, 37°C, 10′ versus mex67-5, 10′ at 25°C = 0.07, pmex67-5, 37°C, 10′ versus mex67-5, 60′ at 25°C = 7.43E-33, pmex67-5, 37°C, 10′ versus mex67-5, 120′ at 25°C = 1.08E-38. (E) HSP104 RNA translational status over the experimental time course analyzed by 35S-methionine pulse labeling of WT (lanes 1–5) or mex67-5 (lanes 6–10) cells. Molecular Cell 2012 48, 145-152DOI: (10.1016/j.molcel.2012.07.022) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Prematurely Released HSP104 RNA Is Not Translated (A) HSP104 RNA-FISH of fixed mex67-5 cells transformed with a SUB2 containing 2μ plasmid, or its vector-only control. RNA-FISH probes were as in Figure 1B. (B) Quantification of cytoplasmic HSP104 RNA levels of strains from (A). Standard deviations of individual experiments were calculated as in Figure 1C. Number of cells counted was as follows: nmex67-5 vector, 37°C 10′ = 131, nmex67-5 pSUB2, 37°C 10′ = 162, nmex67-5 vector, 120′ at 25°C = 120, nmex67-5 vector, 120′ at 25°C = 131. T test: pmex67-5 vector 37°C, 10′ versus mex67-5 pSUB2, 37°C, 10′ = 2.40E-16, pmex67-5 pSUB2, 37°C, 10′ versus mex67-5 vector, 120′ at 25°C = 0.23, pmex67-5 pSUB2, 37°C, 10′ versus mex67-5 vector, 120′ at 25°C + thiolutin = 0.03. (C) HSP104 RNA translational status analysis (as in Figure 1E). Hsp104p levels (black arrow) were normalized to a constant band beneath Hsp104p (red arrow). Average Hsp104p levels with standard deviations were calculated (n = 3). mex67-5pSUB2, 37°C 10′ versus mex67-5vector, 37°C 10′, 60′ at 25°C = 1.8% ± 17.1%, mex67-5pSUB2, 37°C 10′ versus mex67-5vector, 37°C 10′, 120′ at 25°C = 3.1% ± 28.2%. (D) Growth capabilities of WT and mex67-5 cells, containing SUB2 expressing plasmid or empty vector as indicated, were analyzed by plating a 10× dilution series on URA-selective plates for 2 days at the indicated temperatures. Molecular Cell 2012 48, 145-152DOI: (10.1016/j.molcel.2012.07.022) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Co-overexpression of RRP6 Counteracts the Toxicity of Excess SUB2 in mex67-5 Cells (A) Growth capabilities of mex67-5 cells, containing SUB2- and/or RRP6-expressing plasmids, were analyzed as in Figure 2D. (B) RNA-FISH, performed and analyzed as in Figure 1B, of HSP104 in mex67-5 overexpressing SUB2 and/or RRP6 as indicated. (C) Quantification of cytoplasmic HSP104 RNA levels of strains from (B). Standard deviations of individual experiments were calculated as in Figure 1C. Number of cells counted was as follows: nmex67-5 vector vector, 37°C 10′ = 131, nmex67-5 vector pRRP6, 37°C 10′ = 136, nmex67-5 pSUB2 vector, 37°C 10′ = 102, nmex67-5 pSUB2 pRRP6, 37°C 10′ = 134. T test: pmex67-5 vector vector 37°C, 10′ versus mex67-5 vector pRRP6, 37°C, 10′ = 0.003, p mex67-5 vector vector 37°C, 10′ versus mex67-5 pSUB2 vector, 37°C, 10′ =1.49E-13, p mex67-5 vector vector 37°C, 10′ versus mex67-5 pSUB2 pRRP6, 37°C, 10′ =0.024, p mex67-5 pSUB2 vector 37°C, 10′ versus mex67-5 pSUB2 pRRP6, 37°C, 10′ =8.41E-11. (D) RNase H northern blotting analysis using the DL163 cleavage oligo (Figure 4A) together with an oligo d(T)20. Northern membrane was probed for HSP104 RNA with radiolabeled DL164 DNA oligonucleotide (Figure 4A), and SCR1 was probed as loading control. Note that the slightly slower migration of the HSP104 RNA band in the “wt + pSUB2 vector” lane is due to a gel artifact. Molecular Cell 2012 48, 145-152DOI: (10.1016/j.molcel.2012.07.022) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Hyperadenylated HSP104 RNA in mex67-5 Cells Is Deadenylated during the 25°C Chase Period (A) Schematic representation of RNase H/northern assay, using the DL163-cleaving DNA oligonucleotide directed towards the 3′ end of HSP104 RNA and an oligo dT-cleaving oligo directed against the poly(A) tail. Bold dark gray line denotes the 5′ end radiolabeled DNA oligonucleotide DL164 used as a probe. (B) RNase H/DL163 northern analysis of total RNA harvested from WT or mex67-5 cells, containing SUB2 expressing plasmid or empty vector as indicated, and subjected to a 10 min 37°C heat pulse. SCR1 RNA was used as loading control. Poly(A) tail lengths, as measured by size markers (data not shown), are denoted to the left of the image. (C) RNase H/northern analysis as in (B) using total RNA harvested from the indicated strains subjected to a 10 min 37°C heat pulse. (D) RNase H/DL163 northern analysis of total RNA harvested from WT and mex67-5 cells subjected to the experimental pulse-chase outline from Figure 1A. Experimental conditions, loading control, and denotations are as in (B). Lane scans to the right of the image quantify the RNA signals. (E) RNase H/DL163/oligo dT northern analysis of RNA samples from (D). Lane scans as in (D). Molecular Cell 2012 48, 145-152DOI: (10.1016/j.molcel.2012.07.022) Copyright © 2012 Elsevier Inc. Terms and Conditions