Multiple Puf proteins regulate the stability of ribosome biogenesis transcripts

Abstract

Cells must make careful use of the resources available to them. A key area of cellular regulation involves the biogenesis of ribosomes. Transcriptional regulation of ribosome biogenesis factor genes through alterations in histone acetylation has been well studied. This work identifies a post-transcriptional mechanism of ribosome biogenesis regulation by Puf protein control of mRNA stability. Puf proteins are eukaryotic mRNA binding proteins that play regulatory roles in mRNA degradation and translation via association with specific conserved elements in the 3' untranslated region (UTR) of target mRNAs and with degradation and translation factors. We demonstrate that several ribosome biogenesis factor mRNAs in Saccharomyces cerevisiae containing a canonical Puf4p element in their 3' UTRs are destabilized by Puf2p, Puf4, and Puf5p, yet stabilized by Puf1p and Puf3p. In the absence of all Puf proteins, these ribosome biogenesis mRNAs are destabilized by a secondary mechanism involving the same 3' UTR element. Unlike other targets of Puf4p regulation, the decay of these transcripts is not altered by carbon source. Overexpression of Puf4p results in delayed ribosomal RNA processing and altered ribosomal subunit trafficking. These results represent a novel role for Puf proteins in yeast as regulators of ribosome biogenesis transcript stability.

Keywords: 3ʹ UTR; Mrna decay; Puf proteins; Pumilio; mRNA stability; ribosome biogenesis; yeast.

Figure 1.

mRNAs involved in ribosome biogenesis are regulated redundantly by Puf4p and Puf5p. Decay analyses are shown of endogenous mRNAs (a) or reporter mRNA constructs PGK1-ALB1 3ʹ UTR and PGK1-RRS1 3ʹ UTR (b) in wild-type (WT), single puf deletion, double puf4Δ/puf5Δ deletion, puf1-5Δ deletion, and puf1-6Δ strains. Representative Northern blots are in the left panels, with average half-life (T_1/2) listed to the right of each blot, and a graphical representation of the average half-lives in the right panels. Minutes following transcriptional repression at time 0 are indicated above the blots. Error bars represent standard error of the mean (SEM) and are representative of ≥ 3 trials. Asterisks indicate the only significantly different half-life in the group as determined by one-way ANOVA with Tukey’s post-hoc test (p ≤ 0.005).

Figure 2.

Reporter mRNAs are not stabilized in galactose, while Puf protein overexpression rescues rapid decay. Decay analyses are shown of reporter constructs PGK1-ALB1 3ʹ UTR and PGK1-RRS1 3ʹ UTR in (a) the WT strain in the presence of dextrose or galactose, or (b) in the double puf4Δ/puf5Δ deletion strain containing an empty vector (EV) or a vector overexpressing Puf4p or Puf5p. Representative Northern blots are in the left panels, with average half-life (T_1/2) listed to the right of each blot, and a graphical representation of the average half-lives in the right panels. Minutes following transcriptional repression at time 0 are indicated above the blots. Error bars represent SEM (n ≥ 3). The asterisk in (a) indicates a significant difference as determined by Student’s t-Test (p ≤ 0.05). The letters next to the bar graph in (b) indicate significant differences as determined by one-way ANOVA with Tukey’s post-hoc test (PGK1-ALB1, p ≤ 0.005; PGK1-RRS1, p ≤ 0.05).

Figure 3.

Puf2p acts with Puf4p and Puf5p to stimulate mRNA decay, while Puf1p and Puf3p stabilize mRNA targets. Decay analyses are shown of reporter constructs PGK1-ALB1 3ʹUTR and PGK1-RRS1 3ʹ UTR in (a) triple mutant strains or (b) in the puf1-5Δ strain containing an empty vector (EV) or a vector overexpressing the Puf1p repeat domain (Puf1RD) or full length Puf1p (Puf1FL) or the Puf3p repeat domain (Puf3RD) or full length Puf3p (Puf3FL). Representative Northern blots are in the left panels, with average half-life (T_1/2) listed to the right of each blot, and a graphical representation of the average half-lives in the right panels. Minutes following transcriptional repression at time 0 are indicated above the blots. Error bars represent SEM (n ≥ 3). Asterisks in (a) represent the only significantly different half-life as compared to the double puf4Δpuf5Δ half-life as determined by one-way ANOVA with Tukey’s post-hoc test (p ≤ 0.05).

Figure 4.

A single Puf Response Element (PRE) in the 3ʹ UTR is critical for decay regulation. Decay analyses are shown of native PRE reporter constructs PGK1-ALB1 3ʹ UTR and PGK1-RRS1 3ʹ UTR and respective mutant PRE reporter constructs (Mut PRE) in the WT and puf1-5Δ strains. In the mutant PRE constructs, the canonical UGUA portion of the PRE was mutated to ACAC. Representative Northern blots are in the left panels, with average half-life (T_1/2) listed to the right of each blot, and a graphical representation of the average half-lives in the right panels. Minutes following transcriptional repression at time 0 are indicated above the blots. Error bars represent SEM (n ≥ 3).

Figure 5.

Puf4p overexpression causes accumulation of rRNA processing intermediates. (a) Representative gel images are shown of the steady state levels of rRNA processing intermediates/precursors in the WT strain in the presence of a CEN empty vector (CEN EV) or a CEN vector overexpressing Puf4p (Puf4 OE). The scRI loading controls for each set of rRNA species is shown directly underneath the corresponding images. The diagrams to the right of each gel image are visual representations of the sequence regions within the rRNA intermediates/precursors. The three final species of rRNA originating from the PolI transcript (18S, 25S and 5.8S) are labeled in white; sites of processing are labeled in black letters A-D. (b) Graphical representation of the rRNA intermediate/precursor band intensities in (a). Error bars represent SEM (n = 3). Asterisks indicate significant differences between reported levels for EV and 4OE as determined by Student’s t-Test (p ≤ 0.01) (c) Representative gel images of rRNA processing intermediates/precursors following an L-[Methyl-³H]-Methionine pulse-chase labeling assay in a WT strain in the presence of a CEN empty vector (CEN EV) or a CEN vector overexpressing Puf4p (Puf4 OE). Minutes following the addition of unlabeled methionine at time 0 are indicated above the gels (n = 3).

Figure 6.

Puf4p overexpression results in nuclear accumulation of ribosomal subunits. (a) Representative panels are shown from confocal microscopy of cells from the WT strain, or the WT strain containing an empty vector (EV) or a vector overexpressing Puf4p (4OE). Labels above the panels indicate the stain/protein being observed. Arrows on the upper set of panels indicate the position of nuclear foci as detected by RPL11B-eGFP. Arrows on the lower set panels indicate the position of nuclei in the cells as detected by a decreased signal of RPS2-eGFP. (b) Graphical representation of the nuclear to cytoplasmic ratio of GFP fluorescence intensity in WT cells ± empty vector or vector overexpressing Puf4p. Letters a and b indicate significant differences using one-way ANOVA (p < 0.005) and Tukey’s post-hoc test. Error bars represent SEM and are representative of ≥ 250 cells spread across 3 individual growth trials.

Figure 7.

Puf proteins regulate mRNA decay to ensure proper ribosome biogenesis. (a) Puf2p, Puf4p, and Puf5p can each act to stimulate mRNA decay of ribosome biogenesis factors through binding to a single PRE site (UGU). Puf1p and Puf3p act through the same PRE to stabilize these mRNAs, potentially by blocking access of another decay factor to the PRE. In the absence of Puf proteins, a secondary mechanism that also requires the PRE site acts to stimulate decay. (b) At physiological levels of Puf proteins, processing of rRNA transcripts occurs normally. When Puf4p is overexpressed, processing is slowed, resulting in higher levels of the initial 35S and aberrant 23S transcripts, and lower levels of 20S and 7s intermediates. (c) At physiological levels of Puf proteins, ribosomal subunits are shuttled from the nucleus at a normal rate. When Puf4p is overexpressed, trafficking of ribosomal subunits is inhibited, resulting in abnormal nuclear retention of ribosomal subunits.