1. Regulated ARE-Mediated mRNA Decay in Saccharomyces cerevisiae
Shobha Vasudevan, Stuart W. Peltz 
Molecular Cell 
Volume 7, Issue 6, Pages (June 2001)
DOI: /S (01)

2. Figure 1 AU-Rich Sequence Elements
The consensus pentamer and nonamer sequences of an ARE are underlined. MFA2 ARE consists of domain I and domain II (underlined). This element was replaced with the ARE from TNFα and from c-fos or with the 3′-UTR of TIF51A or PGK1as described in Experimental Procedures
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
The AU-Rich 3′-UTR of TIF51A Is Sufficient to Mediate Carbon Source Regulation of mRNA Stability
(A) Y19(rpb1-1) cells were grown at 25°C in minimal media with 2% glycerol or 2% dextrose as the carbon source. Transcription was shutoff by shifting the rpb1-1 strain to the nonpermissive temperature of 37°C and time points taken. Half-life measurements were made as described in Experimental Procedures. Total RNA was extracted and run on a 1% formaldehyde gel followed by northern blotting. The blot was probed with the 3′-UTR of TIF51A.
(B) Strain Y18 (rpb1-1) was used to monitor the effect of carbon source on the endogenous MFA2 transcript. Half-life measurements were made as before. The northern blot obtained was probed for the coding region of MFA2.
(C) The chimeric MFA2-TIF51A construct (p5048) was transformed into Y496 (MFA1::LEU2; MFA2::URA3; rpb1-1). Half-life measurements were made as described . The transformants were either grown at 25°C in minimal media with 2% glucose or with 2% glycerol as the carbon source. Total RNA was extracted and RNase protection analysis performed using antisense probes to the coding region of MFA2 and U3 as a loading control. The gel was quantitated using a Molecular Imager (Molecular Dynamics PSI-PC)
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3 Mammalian AREs Are Regulated by Carbon Source in Yeast Cells
The chimeric MFA2 constructs were transformed into Y496 (MFA1::LEU2; MFA2::URA3; rpb1-1): (A) MFA2-TNFα (p5018); (B) MFA2-c-fos (p5016); (C) MFA2-PGK1 (p5042). Half-life measurements were made as described. The transformants were either grown at 25°C in minimal media with 2% glucose or with 2% glycerol as the carbon source. Transcription was shut off by shifting the rpb1-1 strain to the nonpermissive temperature of 37°C and time points taken. Total RNA was extracted and run on a 1% formaldehyde gel followed by Northern blotting. The blots were probed for the coding region of MFA2. The blots were probed for U3 for normalizing loading error
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Mechanism of Decay of AREs in Yeast
The ARE constructs are under the control of the GAL1 UAS in a CEN plasmid with a poly(G) tract 27 nt upstream of the AUG of the MFA2-coding region. Y19 (rpb1-1) cells were transformed with (A) MFA2-TNFα (p5043), (B) MFA2-TIF51A (p5045), and (C) MFA2-PGK1 (p5044). The transformants were grown in 2% glycerol till they reached an absorbance 600 nm of 0.6. The cells were then induced to express the transcript for 8 min in 3% galactose followed by a temperature shift to 37°C using prewarmed glycerol or glycerol media to shut off transcription of the rpb1-1 strain. Time points were taken and total RNA extracted as described. Twenty micrograms was loaded on a 7 M urea, 6% acrylamide gel, which was blotted and probed for the coding region of MFA2. The intermediates indicated on the side represent the adenylated transcript, the deadenylated transcript, and the decapped intermediate, which is degraded from the 5′ end up to the poly G tract (Muhlrad et al., 1994). The small amount of decapped product is present at the 0 time point as a result of preinduction (data not shown) and accumulates very slowly with time in glucose conditions
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 The Hog1p/p38 Kinase Pathway Regulates ARE-Mediated Decay
(A) Y19 (rpb1-1) cells were transformed with p5055 (CTR1-MFA2-TNFα) and treated with 20 μM of SB for 1 hr. The transformants were grown in 2% glucose till they reached an absorbance 600 nm of 0.6. The cells were then induced to express the transcript for 5 min by adding the Copper chelator. This was followed by addition of copper sulfate and a temperature shift to 37°C using prewarmed glucose media to shut off transcription as described in Experimental Procedures. Twenty micrograms of RNA extracted from each time point was loaded on a 7 M urea, 6% acrylamide gel, which was blotted and probed for the coding region of MFA2 or for the endogenous CYH2 transcript. The intermediates indicated on the side represent the adenylated transcript, the deadenylated transcript, and the decapped intermediate, which is degraded from the 5′ end up to the poly G tract (Muhlrad et al., 1994). The decapped product is present at the zero time point as a result of preinduction (data not shown) and accumulates very slowly with time in glucose conditions.
(B) Strains Y497 (wild-type) and Y508 (hog1Δ) were transformed with p5055 and half-life measurements taken as before except 20 μg/ml of thiolutin was used along with copper sulfate to shut off transcription
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
Pub1p Cross-Links Specifically to the ARE in Glucose and Not in Nonglucose Conditions
(A) Cross-linking analysis with TNFα ARE was performed with both extracts made from Y19 (rpb1-1) cells grown in glycerol and glucose as described in Experimental Procedures. Cold oligo TNFα RNA was synthesized by Integrated DNA Technologies (Coralville, IA) and was added in the following order: 1×, 10×, 50×, 100×. 100× of NSC (nonspecific competitor) is the mutated version of the TNFα ARE; MT-ARE-A60 was also synthesized by Integrated DNA Technologies.
(B) Extracts from isogenic RY262 (wild-type) and Y468 (pub1Δ) cells were made as described and cross-linked to labeled TNFα ARE in the presence or absence of 50× cold ARE competitor or the nonspecific competitor.
(C) Cross-linking analysis was performed as described with Y19 glucose extracts. A set of the reactions were immunoprecipitated with anti-Pub1 antibody or with anti-Flag antibody as described and then run on a 10% SDS-polyacrylamide gel. All gels were dried and visualized on Molecular Imager Phosphorimager (Molecular Dynamics PSI-PC)
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
Pub1p Is Required for TNFα-Mediated Stabilization in Glucose Conditions
RY262 (wild-type) and Y468 (pub1Δ) were transformed with p5046 to express the chimeric MFA2-TNF. Half-life measurements were made in glucose as described. The blots were probed with DNA probes for MFA2-coding region and for U3 as a loading control
Molecular Cell 2001 7, DOI: ( /S (01) )