Article Figures & Data
Figures
- Fig. 1.
A ΔRIP1 strain is deficient in nuclear export of hs mRNA at 42°C. (A) SDS-PAGE of total cellular proteins labeled with [35S]methionine in wild-type (WT) and ΔRIP1 strains treated for 30 min at 25, 37, or 42°C. The positions of the stress-inducible yeast heat shock proteins Hsp104p, Hsp82p, and Hsp70s are shown by arrows. (B and C) Thermotolerance assays. Each row represents serial dilutions of the same culture. The dilution factor is indicated at the bottom of each column. The number of colonies corresponds to the number of cells that have survived the treatment. (B) 42°C thermotolerance. Wild-type and ΔRIP1 strains were either maintained at 25°C or incubated at 42°C for various times, with or without pretreatment at 37°C prior to the 42°C incubation. The three plates show the number of cells that survived the 42°C treatment for 1, 3, and 6 h, respectively. (C) 50°C thermotolerance. Wild-type and ΔRIP1 strains were either maintained at 25°C or pretreated for 30 min at 42°C prior to a 20-min 50°C treatment.
- Fig. 2.
Several poly(A)+ RNA transport mutants show a defect in heat shock protein synthesis at 42°C. SDS-PAGE of total cellular proteins labeled with [35S]-methionine in wild-type (WT) and various mutant strains treated for 30 min at 25 or 42°C is shown. The positions of the stress-inducible yeast heat shock proteins Hsp104p, Hsp82p, and Hsp70s are shown by arrows. (B) Wild-type heat shock protein labeling pattern is restored upon transformation ofrna1-1 with a plasmid carrying the wild-type RNA1gene.
- Fig. 3.
A block in hs mRNA export in ΔRIP1 (A),rna1-1 (B), and prp20-1 (C) strains is reflected in lowered thermotolerance. Yeast cultures were either maintained at 25°C or pretreated at 42°C for 30 min prior to a 20-min 50°C incubation. Each row represents serial dilutions of the same culture (the dilution factors are the same as in Fig. 1C). The number of growing colonies corresponds to the number of cells that survived the treatment. The thermotolerance defect of the strains transformed with empty vector (a) can be reversed (b) upon transformation with pRIPHi (A), pRNA1 (B), or pPRP20 (C).
- Fig. 4.
Stress-induced nuclear accumulation of poly(A)+ RNA and SSA4 mRNA occurs only in the absence of Rip1p. Cy3 in situ hybridization was performed to localize poly(A)+ RNA and SSA4 mRNA in wild-type and ΔRIP1 strains. The cells were maintained at 25 or 42°C for 1 h. DAPI (4′,6′-diamidino-2-phenylindole) staining for the ΔRIP1 strain at 42°C is shown on the right.
- Fig. 5.
Incubation of a ΔRIP1 strain at 42°C causes a decline in the protein labeling pattern. SDS-PAGE of total cellular proteins labeled with [35S]methionine is shown. The positions of the stress-inducible yeast heat shock proteins Hsp104p, Hsp82p, and Hsp70s are shown by arrows. (A) Wild-type, ΔRIP1, and rpb1-1 strains were incubated for various times at 42 or 37°C, followed by a 5-min labeling with [35S]methionine. (B) Enlargement of a portion of the gel in panel A, showing the delayed appearance of heat shock protein bands in the ΔRIP1 strain at 42°C.
- Fig. 6.
Three heat-shock-inducible SSA4-GFP hybrid mRNAs are efficiently exported in a wild-type strain but not a ΔRIP1strain at 42°C. (A, B, and C) Analysis of GFP synthesis by immunoblotting with α-GFP antibody. The position corresponding to GFP is marked by the arrowhead. Lanes marked c are from cultures maintained at 25°C. (A) Time course of GFP expression upon induction of HS-GFP and HS-GFP-3′ SSA4 mRNAs at 42°C in wild-type (WT) and ΔRIP1 strains. (B) Time course of GFP expression upon induction of HS-GFP*SSA4-3′ SSA4 mRNA at 37°C in wild-type and ΔRIP1 strains. (C) Time course of GFP expression upon induction of HS-GFP*SSA4-3′ SSA4 mRNA at 42°C in wild-type and ΔRIP1 strains. (D) Primer extension analysis of HS-GFP*SSA4-3′ SSA4 mRNA induction at 42°C in wild-type and ΔRIP1 strains (the same experiment as in panel C). Denaturing PAGE analysis of primer extension products with GFP- and U2-specific primers is shown. Positions corresponding to HS-GFP*SSA4-3′ SSA4 mRNA and U2 RNA are marked by the upper and lower arrowheads, respectively.
- Fig. 7.
Nuclear export of galactose-inducible β-galactosidase (Gal-LacZ) mRNA is inhibited at 42°C in a ΔRIP1strain but not a wild-type strain. (A, B, and C) LacZ protein was analyzed by immunoblotting it with α-β-galactosidase antibody. The position corresponding to the LacZ band is marked by an arrowhead. (A) Time course of LacZ protein expression upon induction at 22 and 37°C in the wild type (WT). The lanes marked c are from cultures maintained at 22°C in the absence of galactose induction. (B) Time course of LacZ protein expression upon induction at 42°C in wild-type and ΔRIP1 strains. The lanes marked c are from cultures treated at 42°C but in the absence of galactose induction. (C) LacZ protein expression after 2-h incubation of wild-type and ΔRIP1 strains at 22, 37, or 42°C, with or without galactose induction. (D) Time course of induction of Gal-LacZ mRNA at 42°C in wild-type and ΔRIP1 strains (the same experiment as in panel B). Denaturing PAGE analysis of extension products with a LacZ-specific primer is shown. The area occupied by multiple extension products is marked by a square bracket.
Tables
- Table 1.
Yeast strains
Strain Genotype Source W303 MAT a ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 canR1-100 48 W303ΔRIP1 MAT a ΔRIP1::KANr; otherwise isogenic to W303 48 rna1-1 MAT a ura3-52 leu2Δ1 trp1 rna1-1 3 prp20-1 MATα ura3-52 leu2Δ1 trp1Δ63 prp20-1 21 prp20-7 MAT a ura3-52 his3Δ200 tyr1 ade2-1 prp20-7 39 prp20-101 MAT a ura3-52 leu2Δ1 his3Δ200 Gal+ prp20-101 51 ΔYRB2 MAT a ura3 leu2 ade2 trp1 YRB2::HIS3 51 mex67-5 MAT a ade2 leu2 ura3 trp1 MEX67::HIS3 pRS314-TRP1-mex67-5 41 mtr2-9 MAT a ade2 leu2 ura3 trp1 MTR2::HIS3 pRS315-LEU2-mtr2-9 41 xpo1-1 MATα ade2 his3 trp1 ura3 can1 XPO1::LEU2 pKW457-HIS3-xpo1-1 45 PSY580 MAT a ura3-52 trp1Δ63 leu2Δ1 Gal+ 42 pse1-1 MAT a ura3-52 trp1Δ63 leu2Δ1 Gal+pse1-1 42 ΔKAP123 MATα ura3-52 leu2Δ1 KAP123::HIS3 42 ΔSXM1 MAT a ura3-52 leu2Δ1 trp1Δ63 ADE+SXM1::HIS3 42 rat8-2 (or dbp5-2) MAT a leu2Δ1 trp1Δ63 ura3-52 rat8-2 44 gle1-8 MAT a ade2 his3 leu2 trp1 ura3 gle1-8 F. Stutz rat7-1 (or nup159-1) MAT a trp1Δ63 ura3-52 leu2Δ1 rat7-1 10 ΔNUP133 MATα ade2 his3 leu2 trp1 ura3 NUP133::HIS3 5 ΔNUP100 MAT a ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 nup100-3::TRP1 52 ΔNUP116 MAT a ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 nup116-5::HIS3 52 ΔNUP145 MAT a ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 nup145-1::URA3 52 NUP82Δ108 MAT a ade2 leu2 ura3 trp1 NUP82::HIS3 pRS316-URA-NUP82Δ108 16 nup49-313 MATα ade2 ade3 his3 leu2 ura3 NUP49::TRP1 pUN100-LEU2-nup49-313 5 ΔNUP57 MAT a ade2 leu2 ura3 trp1 NUP57::HIS3 E. Hurt, V. Doye rpb1-1 MATα leu2 ura3 Gal+ rpb1-1 R. Young
