Fig 1.
The SDH4 mRNA is translationally repressed under iron deficiency.
(A) Wild-type BY4741 strain transformed with pRS416-Flag2-SDH4 (SDH4) was grown at 30°C for 7 h in SC-Ura with 10 μM FAS (+Fe) or in SC-Ura with 100 μM BPS (-Fe). Flag2-SDH4 and PGK1 mRNA levels were determined by RT-qPCR using specific primers. Flag2-Sdh4 and Pgk1 protein levels were determined by Western blot with anti-Flag and anti-Pgk1 antibodies, respectively. Flag2-SDH4 translation efficiency was calculated as follows: (Flag2-Sdh4 protein / Flag2-SDH4 mRNA) / (Pgk1 protein / PGK1 mRNA). Mean values and standard deviations from at least two independent experiments are shown and referred to those in +Fe. (B) Flag2-Sdh4 (F2-Sdh4)-expressing cells were grown as in (A) and Flag2-Sdh4 protein levels were determined at successive times after adding 50 μg/mL of CHX. A representative experiment is shown. Mean values of F2-Sdh4 protein half-life (t1/2) from two independent experiments are shown. A non-specific anti-Flag band (*) was used as loading control. (C, D, E, and F) sdh4Δ mutant strain transformed with pRS416-SDH4 plasmid (SDH4) was cultivated as mentioned above and polysomal fractionation was carried out as described in Materials and Methods. The A260nm profiles after gradient fractionation in +Fe and -Fe are shown (E and F, respectively), and the ribosomal subunits (40S and 60S), monosomes (80S) and polysomes are indicated. The RNA in individual fractions was extracted and SDH4 (C) and ACT1 (D) mRNA levels were analyzed by RT-qPCR as described in Materials and Methods.
Fig 2.
The SDH4 mRNA AREs are required for its translational repression under iron-deficient conditions.
(A) sdh4Δ cells transformed with plasmids pRS416-Flag2-SDH4 (SDH4) or pRS416-Flag2-SDH4-AREmt (SDH4-AREmt) were grown at 30°C for 7 hours in SC-Ura with 100 μM BPS. Flag2-Sdh4 and Pgk1 protein and mRNA levels were determined by Western blot and RT-qPCR, respectively. Translation efficiency was calculated as in Fig 1A. Mean values and standard deviations from two independent experiments are shown and referred to pRS416-Flag2-SDH4 (SDH4). (B-E) sdh4Δ mutant cells transformed with pRS416-SDH4 (SDH4) or pRS416-SDH4-AREmt (SDH4-AREmt) were grown under iron-deficient conditions as mentioned above (-Fe: B and C) or in SC-Ura (+Fe; D and E). Polysomal fractionation and analysis of SDH4 (B, D) and ACT1 (C, E) mRNA levels were carried out as described in Materials and Methods.
Fig 3.
Cth2 represses SDH4 mRNA translation in a Cth2-TZF-dependent manner when iron is scarce.
(A) cth1Δcth2Δ mutant cells co-transformed with plasmids pRS416-Flag2-SDH4 and either pRS415-CTH2 (CTH2), pRS415 (cth2Δ) or pRS415-CTH2-C190R (CTH2-C190R) were grown at 30°C for 7 h in SC-Ura-Leu supplemented with 100 μM BPS. Flag2-Sdh4 and Pgk1 protein/mRNA ratios were determined by Western blot/RT-qPCR, respectively, and translation efficiency was calculated as in Fig 1A. Mean values and standard deviation of three independent biological replicates are shown and referred to pRS415-CTH2 (CTH2). (B and C) Yeast cth1Δcth2Δsdh4Δ mutant cells co-transformed with plasmids pRS416-SDH4 and pRS415-CTH2 (CTH2) or pRS415 (cth2Δ) were cultivated as mentioned above and polysomal fractionation was carried out as described in Materials and Methods. The RNA in individual fractions was extracted and SDH4 (B) and ACT1 (C) mRNA levels were analyzed by RT-qPCR as described. (D and E) Yeast cth1Δcth2Δsdh4Δ mutant cells co-transformed with plasmids pRS416-SDH4 and pRS415-CTH2 (CTH2) or pRS415-CTH2-C190R (CTH2-C190R) were grown under iron-deficient conditions. Polysomal fractionation and RT-qPCR analysis were performed as above. Representative data from at least two independent experiments are shown.
Fig 4.
CTH2 mRNA is translationally repressed by its ARE in low iron conditions.
(A) cth1Δcth2Δ mutant cells transformed with pRS416-Flag2-CTH2 (CTH2) or pRS416-Flag2-CTH2-AREmt (CTH2-AREmt) were grown at 30°C for 7 hours in SC-Ura with 100 μM BPS (-Fe). Flag2-Cth2 and Pgk1 protein and mRNA levels were determined by Western blot and RT-qPCR, respectively. Flag2-CTH2 translation efficiency was calculated as follows: (Flag2-Cth2 protein / Flag2-CTH2 mRNA) / (Pgk1 protein / PGK1 mRNA). Mean values and standard deviation from at least two independent experiments are shown and refer to pRS416-Flag2-CTH2 (CTH2). (B and C) cth1Δcth2Δ mutant cells transformed with pRS416-CTH2 (CTH2) or pRS416-CTH2-AREmt (CTH2-AREmt) were cultivated as mentioned above and polysomal fractionation was carried out as described in Materials and Methods. The RNA in individual fractions was extracted and percentages of CTH2 (B) and ACT1 mRNA (C) were analyzed by RT-qPCR as described in Materials and Methods. Representative data from at least two independent experiments are shown.
Fig 5.
Cth2 represses its own mRNA translation in a Cth2-TZF-dependent manner under iron-limited conditions.
(A) cth1Δcth2Δ mutant cells transformed with plasmids pRS416-Flag2-CTH2 (CTH2) or pRS416-Flag2-CTH2-C190R (CTH2-C190R) were grown at 30°C for 7 hours in SC-Ura with 100 μM BPS (-Fe). Mean values and standard deviation from four independent experiments of steady-state mRNA, proteins and CTH2 translation efficiencies were determined and normalized as in Fig 4A. (B and C) cth1Δcth2Δ mutant cells transformed with pRS416-CTH2 (CTH2) or pRS416-CTH2-C190R (CTH2-C190R) were cultivated as mentioned above. Polysomal fractionation was carried out as described in Materials and Methods. The RNA of individual fractions was extracted and the percentages of CTH2 (B) and ACT1 mRNA (C) were analyzed by RT-qPCR as described in Materials and Methods. Representative data from at least two independent experiments are shown.
Fig 6.
Cth2 represses the translation of other ARE-containing mRNAs under iron-limited conditions.
cth1Δcth2Δsdh4Δ mutant strains co-transformed with pRS416-SDH4 and either pRS415-CTH2 (CTH2) or pRS415 (cth2Δ) plasmids were cultivated at 30°C for 7 h in SC-Ura-Leu with 100 μM BPS (-Fe). Polysomal fractionation was carried out as described in Materials and Methods. The percentages of endogenous CCP1 (A), HEM15 (B), WTM1 (C) and ACT1 (D) mRNAs from unified monosomal and polysomal fractions were determined by RT-qPCR as described in Materials and Methods. Mean values and standard deviations from three independent experiments are shown.
Fig 7.
Both Cth2 NTD and CTD are necessary for SDH4 mRNA translational repression, whereas only Cth2 NTD is implicated in SDH4 mRNA decay upon iron starvation.
(A) Schematic representation of the GFP-CTH2 fusion and truncations made at the NTD (ΔN) and CTD (ΔC) of Cth2 protein. (B) cth1Δcth2Δ mutant cells co-transformed with plasmids pRS415-Flag2-SDH4 and either pRS416-GFP-CTH2 (CTH2), pRS416 (cth2Δ), pRS416-GFP-CTH2ΔN89 (CTH2ΔN89), pRS416-GFP-CTH2ΔN170 (CTH2ΔN170) or pRS416-GFP-CTH2ΔC52 (CTH2ΔC52) were cultivated at 30°C for 7 h in SC-Ura-Leu with 100 μM BPS. Flag2-Sdh4 and Pgk1 protein and mRNA levels were determined by Western blot and RT-qPCR, respectively. Translation efficiency was calculated as in Fig 1A. Mean values and standard deviations from three independent experiments are shown and refer to pRS416-GFP-CTH2 (CTH2). An asterisk (*) indicates a significant p-value (≤ 0.03) compared with CTH2. (C) cth1Δcth2Δsdh4Δ mutant cells co-transformed with plasmids pRS415-SDH4 and either pRS416-GFP-CTH2 (CTH2), pRS416 (cth2Δ) or pRS416-GFP-CTH2ΔN170 (CTH2ΔN170) were cultivated at 30°C for 7 h in SC-Ura-Leu with 100 μM BPS. Polysomal fractionation was carried out as described in Materials and Methods. The RNA in individual fractions was extracted and the percentages of SDH4 mRNA were analyzed by RT-qPCR as described in Materials and Methods. Representative data from at least two independent experiments are shown. (D) cth1Δcth2Δsdh4Δ mutant cells co-transformed with plasmids pRS415-GAL1-SDH4 and either pRS416-GFP-CTH2 (CTH2), pRS416 (cth2Δ) or pRS416-GFP-CTH2ΔC52 (CTH2ΔC52) were cultivated as described in Materials and Methods for the determination of SDH4 mRNA half-life. Mean values and standard deviations from three independent experiments are shown. The asterisk (*) indicates a significant p-value (≤ 0.04) compared with cth2Δ. (E) cth1Δcth2Δsdh4Δ mutant cells co-transformed with plasmids pRS415-SDH4 and either pRS416-GFP-CTH2 (CTH2), pRS416 (cth2Δ) or pRS416-GFP-CTH2ΔC52 (CTH2ΔC52) were cultivated and analyzed as in panel C. Representative data from at least two independent experiments are shown.
Fig 8.
The Cth2 CTD mutant shows growth defects under iron-deficient conditions.
(A) cth1Δcth2Δ mutant cells transformed with plasmids pRS416-GFP-CTH2 (CTH2), pRS416 (cth2Δ) or pRS416-GFP-CTH2ΔC52 (CTH2ΔC52) were grown to the exponential phase and spotted in 10-fold serial dilutions on SC-Ura and SC-Ura with 700 μM Ferrozine plates as described in Materials and Methods. (B and D) cth1Δcth2Δ mutant cells transformed with plasmids pRS416-GFP-CTH2 (CTH2), pRS416 (cth2Δ) or pRS416-GFP-CTH2ΔC52 (CTH2ΔC52) were inoculated at an OD600nm of 0.1 in SC-Ura (B) and SC-Ura with 700 μM Ferrozine (D) and growth was determined by OD600nm measurements as described in Materials and Methods. The growth curve values represented are the mean of three independent biological samples. (C and E) μmax values for the growth curves represented in B (C) and D (E) were calculated as described in Materials and Methods. Mean values and standard deviations from at least three independent experiments are shown and referred to the value of CTH2-expressing cells in +Fe. The asterisk (*) indicates a significant p-value (≤ 0.02) compared with CTH2 in -Fe.