Multiple RNA Surveillance Pathways Limit Aberrant Expression of Iron Uptake mRNAs and Prevent Iron Toxicity in S. cerevisiae  Albert Lee, Anthony K. Henras, Guillaume Chanfreau  Molecular Cell  Volume 19, Issue 1, Pages 39-51 (July 2005) DOI: 10.1016/j.molcel.2005.05.021 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Altered Iron Uptake mRNAs Metabolism in the RNase III Knockout Strain (A) Northern analysis of a lacZ mRNA (diagrammed) under the control of an iron-regulated promoter in wild-type (wt) and rnt1Δ strains. Strains were grown in minimal medium lacking uracil with normal iron concentrations. (B) Northern analysis of iron uptake mRNAs. Strains were grown in minimal medium with normal iron concentrations. (C) Northern analysis of iron uptake mRNAs in wt, rnt1Δ strain, and a strain expressing a catalytically inactive mutant, E320K. Cells were shifted for 4 hr in media containing BPS and the indicated iron concentrations. (D) The iron starvation response is delayed in RNase III-deficient strain. RNA samples were extracted from wt or rnt1Δ strains after the indicated times after shift into iron-depleted medium. Molecular Cell 2005 19, 39-51DOI: (10.1016/j.molcel.2005.05.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Mislocalization of Rnt1p in the Cytoplasm Inhibits the Iron Starvation Response (A) Northern blot analysis of RNAs extracted from a rnt1Δ strain transformed with the pUG35-RNT1 plasmid expressing wt Rnt1p fused to GFP or with the pUG35-RNT1ΔC11 expressing a truncated version of Rnt1p lacking the C-terminal nuclear localization signal. Legend as in Figure 1. (B) Quantification of the Northern blots signals. Values are averages from triplicate experiments. Error bars indicate the SD. For each mRNA, the values were divided by the value obtained from the average levels observed in the rnt1Δ strain transformed with the pUG35-RNT1 plasmid, divided by the same ratio for the PDC1 internal control. Molecular Cell 2005 19, 39-51DOI: (10.1016/j.molcel.2005.05.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Stem-Loop Structures Typical of Rnt1p Substrates Are Present in Many Iron Uptake mRNAs (A) Theoretical folds obtained by systematic folding of the corresponding RNAs by using Mfold (Zuker, 2003). The regions indicated for each gene are: ARN2 #1, 1137–1179; ARN2 #2, 638–723; ARN3, 1420–1517; ARN4, 1601–1650; FIT1, 1320–1369; FIT2, −221–126; FIT3, 375–430; FRE2, 2096–2135; FRE3 #1, 1043–1110; and FRE3 #2, 115–185. Numberings are according to the A of the AUG initiation codons. The stop codon of FRE2 is boxed. (B) In vitro cleavage of model ARN2 and FIT2 substrates. In vitro transcripts containing the regions indicated on (A) for ARN2 and FIT2 were incubated with buffer (lanes 1), wt Rnt1p (lanes 3), or E320K mutant (lanes 2). Primer extensions were performed to map the cleavage sites. A sequencing ladder was generated by using the same 5′-labeled primer and run in parallel. (C) Effect of the FIT1 stem-loop deletion on the FIT1 mRNA levels. Northern blot was performed on a wt strain or on two strains carrying a deletion of the FIT1 stem loop (indicated ΔSL in [A]) shifted in low iron conditions for 210 min. Values are the average of triplicate experiments (wt) and of duplicate experiments performed on the two independent mutant clones (ΔSL) standardized to the wt. Error bars represent the SD. Molecular Cell 2005 19, 39-51DOI: (10.1016/j.molcel.2005.05.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Analysis of the Expression of Iron mRNAs in Exonuclease Mutant Strains (A) Analysis of the expression of iron uptake mRNAs in cells lacking 5′→3′ exonucleases activities or the Rrp6p component of the nuclear exosome. Membranes were hybridized with the probes corresponding to the different mRNAs. Also shown are ethidium bromide staining of agarose gels (EtBr). (B) Expression of the FIT3 gene in wt and ribonuclease mutant strains. (C) RNase H mapping of the 5′-extended forms of FIT3. RNAs extracted from an xrn1Δ rat1-1 strain grown at 25°C were analyzed by RNase H and Northern blot by using the corresponding oligonucleotides. Numbers correspond to the position of the oliogonucleotide 5′ end (number of nucleotides upstream from the FIT3 ATG). An RNA sample from the rnt1Δ strain was included as a control for the size of the mature FIT3 mRNA. The membrane was hybridized with a probe hybridizing to the FIT3 mRNA. Therefore, only the downstream RNase H cleavage products are detected. (D) Primer extension mapping of the 5′-extended forms of FIT3. Shown is the primer extension product obtained from RNAs extracted from the xrn1Δ rat1-1 strain grown at 25°C. Molecular Cell 2005 19, 39-51DOI: (10.1016/j.molcel.2005.05.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Rnt1p and the 5′→3′ Exonucleases Xrn1p and Rat1p Cooperate in the Surveillance of Extended Species of the ARN2 and ARN3 mRNAs (A) Expression of the ARN2 mRNA in iron-replete conditions in the rnt1Δ strain and in 5′→3′ exonuclease mutant strains. Cells were grown in iron-replete medium at the indicated temperatures. Northern analysis was performed by using a probe hybridizing to the ARN2 ORF. (B) Expression of the ARN2 mRNA in low iron conditions in wt and rnt1Δ strains. Shown is the structure of the ARN2-Ty5 genomic region and the positions of the probes used for detection. Two different membranes generated from identical samples run on the same gel were hybridized to a probe hybridizing to the ARN2 ORF (#1, left) or to a probe hybridizing to the Ty5-LTR (#2, right). (C) Analysis of the expression of the ARN3 and HPA3 mRNAs in iron-replete conditions in the rnt1Δ strain and in 5′→3′ and rrp6Δ exonuclease mutants. Shown is the genomic structure of the HPA3 and ARN3 loci. Different membranes generated from identical samples run on the same agarose gel were hybridized to an ARN3 probe or an HPA3 probe. Identification of the species was based on their estimated size and on their pattern of hybridization with the two probes and to probes hybridizing to the region upstream from HPA3 or between HPA3 and ARN3 (data not shown). The putative cleavage intermediate is indicated by an arrowhead. The HPA3 species that comigrate with these intermediates are indicated by an asterisk. Molecular Cell 2005 19, 39-51DOI: (10.1016/j.molcel.2005.05.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Cells Lacking RNase III Activity Are Hypersensitive to Iron Toxicity Shown are isogenic wt or rnt1Δ cells grown for seven days at 25°C in synthetic dextrose minimal medium supplemented with the indicated iron or NaCl concentrations. Molecular Cell 2005 19, 39-51DOI: (10.1016/j.molcel.2005.05.021) Copyright © 2005 Elsevier Inc. Terms and Conditions