Conditional regulation of Puf1p, Puf4p, and Puf5p activity alters YHB1 mRNA stability for a rapid response to toxic nitric oxide stress in yeast

Abstract

Puf proteins regulate mRNA degradation and translation through interactions with 3' untranslated regions (UTRs). Such regulation provides an efficient method to rapidly alter protein production during cellular stress. YHB1 encodes the only protein to detoxify nitric oxide in yeast. Here we show that YHB1 mRNA is destabilized by Puf1p, Puf4p, and Puf5p through two overlapping Puf recognition elements (PREs) in the YHB1 3' UTR. Overexpression of any of the three Pufs is sufficient to fully rescue wild-type decay in the absence of other Pufs, and overexpression of Puf4p or Puf5p can enhance the rate of wild-type decay. YHB1 mRNA decay stimulation by Puf proteins is also responsive to cellular stress. YHB1 mRNA is stabilized in galactose and high culture density, indicating inactivation of the Puf proteins. This condition-specific inactivation of Pufs is overcome by Puf overexpression, and Puf4p/Puf5p overexpression during nitric oxide exposure reduces the steady-state level of endogenous YHB1 mRNA, resulting in slow growth. Puf inactivation is not a result of altered expression or localization. Puf1p and Puf4p can bind target mRNA in inactivating conditions; however, Puf5p binding is reduced. This work demonstrates how multiple Puf proteins coordinately regulate YHB1 mRNA to protect cells from nitric oxide stress.

FIGURE 1:

Puf1p, Puf4p, and Puf5p regulate YHB1 mRNA stability. (A) Sequence of the YHB1 3′ UTR. Canonical binding sequences of Puf proteins are in bold. Underlined regions are candidate sites of Puf interaction (site #1 and #2). UGUN sequences are shaded gray. (B) Decay of PGK1/YHB1 3′ UTR fusion mRNA in wild-type (WT), individual PUF deletion, and Δpuf1-5 yeast strains grown to an OD₆₀₀ of 1.0 in galactose, then transcription repressed at time 0 with addition of dextrose. Left, representative Northern blots, with average half-life (T_1/2) listed to the right of each blot. Right, graphical representation of T_1/2. Minutes after transcriptional shut-off are indicated above blots and along the x-axis. The error for each T_1/2 is the SEM (n ≥ 3).

FIGURE 2:

A single, flexible Puf recognition element in the YHB1 3′ UTR is required for Puf1p, Puf4p, and Puf5p regulated decay and binding. (A) Sequence of the Puf recognition element (bolded and underlined) in wild-type YHB1 3′ UTR. Left, mutation of the first UGU to ACU, site #1 mutant, (yhb1-1); right, mutation of both UGU elements to ACA, site #2 mutant, (yhb1-2). Mutated sequences are boxed. (B) Decay of WT, yhb1-1 mutant, and yhb1-2 mutant PGK1/YHB1 3′ UTR fusion mRNA in WT yeast or WT PGK1/YHB1 3′ UTR in Δpuf1-5 yeast. Left, representative Northern blots, with average half-life (T_1/2) listed to the right of each blot. Right, graphical representation of T_1/2. Minutes after transcriptional shut-off are indicated above blots and along the x-axis. The error for each T_1/2 is the SEM (n ≥ 3). (C) Representative gel mobility shift assay of the WT YHB1, yhb1-1 mutant, or yhb1-2 mutant RNA sequence shown in A in the presence or absence of 1.5 μM GST-Puf1p, GST-Puf4p, or GST-Puf5p. Positions of free radiolabeled RNA and RNA bound to a Puf protein (Pufp + RNA) are indicated. (D) Graphical representation of the data from C, with relative levels of Pufp + RNA complexes for each Puf protein on the y-axis. Data are an average of two experiments. (E) Graphical representation of gel mobility shift assays in which increasing concentrations of Puf proteins were incubated with either WT RNA (top) or yhb1-1 mutant RNA (bottom). Relative levels of Pufp + RNA complexes are indicated on the y-axis, with Puf protein concentration on the x-axis. Data are an average of two experiments.

FIGURE 3:

Overexpression of Puf4p or Puf5p in WT yeast stimulates a more rapid decay of target mRNAs. (A) Decay of WT PGK1/YHB1 3′ UTR fusion mRNA in WT yeast coexpressed with empty vector (EV CEN or EV 2 μ), Puf1 full-length (FL), Puf1 repeat domain (RD), Puf4FL, Puf4RD, Puf4FL/CEN, Puf5FL, Puf5RD, or Puf5RD mutant (mut). Left, representative northern blots, with average half-life (T_1/2) listed to the right of each blot. Right, graphical representation of T_1/2. Minutes after transcriptional shut-off are indicated above blots and along the x-axis. Overexpression (OE) constructs used are indicated to the left of the blots. (B) Decay of WT PGK1/YHB1, site #1 mutant (yhb1-1), or site #2 mutant (yhb1-2) in the presence of Puf1FL, Puf4FL/CEN, or Puf5FL overexpression. (C) Decay of PGK1/HXK1 3′ UTR fusion mRNA in the absence (–) or presence of Puf4FL/CEN. (D) Decay of PGK1/PGK1 3′ UTR fusion mRNA in the absence (–) or presence of Puf4FL/CEN. For A, B, and D, the error for each T_1/2 is the SEM (n ≥ 3). For C, the error for each T_1/2 is the SEM (n = 2).

FIGURE 4:

Expression of Puf1p, Puf4p, or Puf5p or the corresponding repeat domain (RD) is sufficient to rescue decay of YHB1 mRNA in a Δpuf1-5 strain. (A) Decay of WT PGK1/YHB1 fusion mRNA in a Δpuf1-5 strain coexpressed with EV 2 μ, Puf1FL, Puf1RD, Puf4RD, Puf5FL, or Puf5RD. Left, representative northern blots, with average half-life (T_1/2) listed to the right of each blot. Right, graphical representation of T_1/2. Minutes after transcriptional shut-off are indicated above blots and along the x-axis. Overexpression (OE) constructs used are indicated to the left of the blots. (B) Decay of WT PGK1/YHB1 fusion mRNA in a Δpuf1-5 strain coexpressed with EV CEN or Puf4FL/CEN. For A, the error for each T_1/2 is the SEM (n ≥ 3) except for EV 2 μ, for which T_1/2 is the SEM (n = 2). For B, the error for Puf4FL/CEN T_1/2 is the SEM (n ≥ 3) and the error for EV CEN T_1/2 is the SEM (n = 2).

FIGURE 5:

Regulation of YHB1 mRNA stability by multiple Puf proteins is dependent on environmental conditions. Decay of PGK1/YHB1 3′ UTR fusion mRNA (A), PGK1/HXK1 3′ UTR fusion mRNA (B), or MFA2/MFA2pG 3′ UTR fusion mRNA (C) in cultures grown in dextrose or galactose carbon sources. Decay of PGK1/YHB1 3′ UTR fusion mRNA (D) or MFA2/MFA2pG 3′ UTR fusion mRNA (E) grown to different final culture densities. Left, representative Northern blots, with average T_1/2 listed to the right of each blot. Right, graphical representation of T_1/2. Minutes after transcriptional shut-off are indicated above blots and along the x-axis. For A–E, the error for each T_1/2 is the SEM (n ≥ 3).

FIGURE 6:

Overexpression of Puf proteins bypasses conditional regulation of YHB1 mRNA stability. Decay of PGK1/YHB1 3′ UTR fusion mRNA (A) or PGK1/HXK1 3′ UTR fusion mRNA (B) in galactose in the absence or presence of Puf4FL/CEN overexpression. Left, representative Northern blots, with average T_1/2 listed to the right of each blot. Right, graphical representation of T_1/2 . Minutes after transcriptional shut-off are indicated above blots and along the x-axis. (C) Decay of PGK1/YHB1 3′ UTR fusion mRNA at OD₆₀₀ of 2 in the absence or presence of Puf4FL/CEN overexpression. (D) Left, percentage cell growth of WT yeast expressing EV CEN, Puf4FL/CEN, or Puf5RD mut or yhb1Δ yeast 24 h after exposure to 3 mM DETA-NO in galactose conditions. Percentage growth is normalized to the growth of strains in the absence of DETA-NO. Right, Northern blot analysis of endogenous, steady-state YHB1 mRNA levels in WT yeast expressing EV CEN or Puf4FL/CEN or puf4Δ or Δpuf1-5 yeast. Levels of YHB1 mRNA relative to the level in WT yeast expressing EV CEN and normalized for loading against scRI RNA are shown below each lane. (E) Decay of endogenous YHB1 mRNA. Representative northern blots are shown, with the average T_1/2 listed to the right of each blot. Yeast strain and expression vectors are indicated to the left of the blots. Minutes after transcriptional shut-off are indicated above the blots. For A–C, the error for each T_1/2 is the SEM (n ≥ 3). For D, the error is the SEM (n ≥ 3). For E, the error for each T_1/2 is the SEM (n ≥ 3).

FIGURE 7:

Effects of carbon source on Puf protein expression level, localization, and mRNA binding. (A) Representative Western blot of Puf1p-TAP, Puf4p-TAP, or Puf5p-TAP levels in cultures grown in YEP media with 2% dextrose or galactose. GAPDH was detected as a loading control for all analyses. (B) Yeast expressing endogenously GFP-tagged Puf1p, Puf4p, and Puf5p were grown in YEP media with 2% dextrose or galactose. Each image represents 10 flattened Z-plane slices through fixed cells using confocal microscopy. Puf1-GFP, Puf4-GFP, and Puf5-GFP are shown in green. Differential image contrasts (DICs) are shown for reference. Bar, equals 5 μm. (C) Endogenously TAP-tagged Puf1p, Puf4p, and Puf5p were immunoprecipitated from yeast grown in YEP medium with 2% dextrose (red bars) or galactose (blue bars). C_q values after IP were compared with no–reverse transcriptase (–RT) C_q values for each mRNA to acquire fold enrichment (top graphs). C_q values after IP in dextrose were compared with C_q values after IP in galactose and normalized to protein levels after IP to acquire mRNA levels bound after IP (bottom graphs). (D) Representative Western blot of protein levels after conditional IPs. Numbers below blots represent relative levels of protein after normalization. For A and B, experiments were done in triplicate. For C and D, experiments were done in duplicate.

FIGURE 8:

Model for Puf protein activity in the presence or absence of environmental stress. During exposure to environmental stresses, including high cell density, NO, and galactose, unknown signaling pathways inactivate Puf1p, Puf4p, and Puf5p, resulting in up-regulation of YHB1 mRNA levels and protein production, allowing for protection from toxic NO levels. In addition, Puf3p activity is inhibited during exposure to galactose, resulting in increased mRNA stability of COX17 mRNA and other mRNAs involved in mitochondrial development and function to accommodate the respiration demand. In the absence of stress, Puf proteins are active and repress target mRNAs.