Carbon source-dependent alteration of Puf3p activity mediates rapid changes in the stabilities of mRNAs involved in mitochondrial function

Abstract

The Puf family of RNA-binding proteins regulates gene expression primarily by interacting with the 3' untranslated region (3' UTR) of targeted mRNAs and inhibiting translation and/or stimulating decay. Physical association and computational analyses of yeast Puf3p identified >150 potential mRNA targets involved in mitochondrial function. However, only COX17 has been established as a target of Puf3p-mediated deadenylation and decapping. We have identified 10 new targets that are rapidly degraded in a Puf3p-dependent manner. We also observed changes in Puf3p activity in response to environmental conditions. Puf3p promotes rapid degradation of mRNA targets in the fermentable carbon source dextrose. However, Puf3p-mediated decay activity is inhibited in carbon sources that require mitochondrial function for efficient cell growth. In addition, the activity of Puf3p is rapidly altered by changing the carbon source. PUF3 expression is not decreased at the RNA or protein level by different carbon sources and localization is not significantly altered, suggesting that Puf3p activity is regulated posttranslationally. Finally, under conditions when Puf3p is unable to stimulate decay, Puf3p can still bind its target mRNAs. Together, these experiments provide insight into the carbon source-specific control of Puf3p activity and how such alterations allow Puf3p to dynamically regulate mitochondrial function.

Figure 1.

Puf3p promotes rapid decay of multiple mRNA targets. (A–M) Representative northern blot analyses of the decay of each mRNA from a WT strain or a puf3Δ strain are shown. Minutes following transcriptional repression are indicated above each set of blots, with the half-lives (T_1/2) and standard error of the mean (SEM) determined from multiple experiments.

Figure 2.

The CYT2 3′ UTR is sufficient to promote Puf3p-mediated mRNA decay, and the conserved UGUA element is required for Puf3p regulation. (A) CYT2 3′ UTR sequences immediately downstream of the translational stop codon for the WT MFA2/CYT2 and mutant MFA2/CYT2mut mRNAs. The conserved 10 nt Puf3p element is highlighted in bold text, with the UGUA element underlined in the WT transcript. In the mutant transcript, the UGUA element is mutated to ACAC, which is boxed. (B) Representative northern blots of the decay of MFA2, MFA2/CYT2 and MFA2/CYT2mut mRNAs expressed from a WT strain or a puf3Δ strain are shown. Minutes following transcriptional repression are indicated above each set of blots, with the half-lives (T_1/2) and standard error of the mean (SEM) determined from multiple experiments.

Figure 3.

Puf3p promotes rapid deadenylation and decay of CYT2 mRNA. Shown are representative northern blot analyses of transcriptional pulse-chase experiments examining decay of CYT2 mRNA from a WT (A) puf3Δ (B) or ccr4Δ strain (C). Minutes following transcriptional repression are indicated above each blot. All mRNAs were cleaved just downstream of the stop codon via oligo-directed RNaseH reactions before gel loading. The 0dT lane in each blot corresponds to mRNA from the 0 min time point in which the poly(A) tail was removed by RNaseH cleavage with oligo(dT). The −8 lane in each blot corresponds to background levels of mRNA expression before galactose induction of CYT2. Size markers (lanes M) are given in nucleotides. The top brackets in each panel (#1 brackets in A and B, sole bracket in C) correspond to mRNAs with full-length 3′ UTRs and poly(A) tail lengths ranging from 0 to 70 adenosines. The #2 brackets in A and B encompass either alternatively cleaved and polyadenylated 3′ UTR species, or transcripts that are both deadenylated and trimmed into the 3′ UTR (top of bracket #2) and species resulting from an additional cleavage product of RNaseH in the 0dT lanes resulting from a poly(A) tract located within the CYT2 3′ UTR (bottom of bracket #2).

Figure 4.

Puf3p-mediated decay of CYT2 mRNA is conditionally regulated. Shown are representative northern blot analyses of the decay of the CYT2 transcript from a WT and a puf3Δ strain grown in media supplemented with dextrose (Dex), ethanol (EtOH), galactose (Gal) or raffinose (Raff). Minutes following transcriptional repression are indicated above each set of blots, with the half-lives (T_1/2) and standard error of the mean (SEM) determined from multiple experiments shown graphically to the right of each set of blots.

Figure 5.

Puf3p-mediated decay of TUF1 mRNA is conditionally regulated. Shown are representative northern blot analyses of the decay of the TUF1 transcript from a WT strain and a puf3Δ strain grown in media supplemented with dextrose (Dex), ethanol (EtOH), galactose (Gal) or raffinose (Raff). Minutes following transcriptional repression are indicated above each set of blots, with the half-lives (T_1/2) and standard error of the mean (SEM) determined from multiple experiments shown graphically to the right of each set of blots.

Figure 6.

Puf3p-mediated decay of COX17 mRNA is conditionally regulated. Shown are representative northern blot analyses of the decay of COX17 (A–C) and STE3 (D) transcripts from a WT strain and a puf3Δ strain grown in media supplemented with dextrose (Dex), ethanol (EtOH), galactose (Gal) or raffinose (Raff). Minutes following transcriptional repression are indicated above each set of blots, with the half-lives (T_1/2) and standard error of the mean (SEM) determined from multiple experiments shown graphically to the right of each set of blots. The average half-life of STE3 mRNA is represented graphically with SEM determined from multiple experiments.

Figure 7.

Puf3p-mediated decay is rapidly activated or inactivated by altering the available carbon source. Shown are graphical representations of the average CYT2 and TUF1 mRNA half-lives as determined by multiple steady-state transcriptional repression experiments. For Puf3p activating conditions (A–C), WT cells were grown in raffinose (Raff) or galactose (Gal) conditions, then briefly incubated in raffinose, galactose or dextrose (Dex) for 2 or 10 min. For Puf3p inactivating conditions (D–F), WT cells were grown in dextrose conditions, then briefly incubated in dextrose, raffinose or galactose for 2 or 10 min. To control for possible alterations to mRNA stabilities due to the resuspension procedure, Raff-Raff, Gal-Gal and Dex-Dex experiments were performed in cells grown continuously in raffinose, galactose or dextrose conditions, harvested, and then incubated with the respective carbon source for 2 min.

Figure 8.

Ethanol, galactose and raffinose do not decrease PUF3 expression levels or alter localization. (A) Northern blot analysis of PUF3 mRNA levels from WT cells grown in YEP media supplemented with 2% dextrose, ethanol, galactose or raffinose is shown with normalized fold changes in expression levels relative to dextrose indicated, along with ethidium bromide staining of 28S and 18S rRNA. (B) Puf3p from WT cells grown in YEP media supplemented with 2% dextrose, ethanol, galactose and raffinose was visualized in the top panel by western blot analysis using antibodies against Puf3p. Western blots were stripped and Tfp1p was visualized in the second panel using antibodies against Tfp1p. Equal OD₆₀₀ units of cells before preparation of protein extracts were calculated for loading onto SDS polyacrylamide gels. Total protein loading was visualized in the bottom panel by Ponceau S staining. (C) Yeast expressing endogenously GFP-tagged Puf3p were grown in YEP media supplemented with 2% dextrose, galactose or ethanol. Each image represents 10 flattened Z plane slices through fixed cells using a confocal fluorescence microsope. Puf3p-GFP is shown in green, while mitochondria stained with Mitotracker Deep Red are shown in red. Merge indicates merger of the Puf3p-GFP and Mitotracker windows. Bright field images of each cell field are shown. The bar equals 5 µm. Fluorescence levels of Puf3p-GFP, quantified across cell fields and normalized to levels in dextrose, are graphically represented.

Figure 9.

Puf3p binds its target mRNAs in both activating and inactivating conditions. Endogenously TAP-tagged Puf3p was immunoprecipitated (IP) from yeast grown in YEP media supplemented with 2% dextrose or galactose using IgG-Sepharose, and RNAs associated with Puf3p were analyzed by qPCR. Fold mRNA enrichment after IP from dextrose (A) or galactose (B) conditions was calculated by comparing average Cq values of mRNAs isolated after IP versus average C_q values of the respective mRNAs isolated from total cell lysate before IP. (C) Average C_q values of mRNAs isolated after IP were compared between dextrose and galactose conditions, with levels normalized to that found in dextrose. (D) Puf3p-TAP protein levels from dextrose and galactose conditions in total cell lysates (Input) and after IP were assessed by western blot using anti-TAP antibodies. A strain expressing nontagged Puf3p (-lane) is also shown.