Fission Yeast PUF Proteins Puf3 and Puf4 Are Novel Regulators of PI4P5K Signaling

Abstract

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3⁺ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3⁺ and puf4⁺, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3⁺ and puf4⁺ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.

INTRODUCTION

Phosphatidylinositol metabolism is an essential signal transduction mechanism to produce important second messengers such as diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃) in eukaryotes. DAG and IP₃ directly or indirectly activates several signal factors such as protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signaling and Ca²⁺-calmodulin-dependent protein kinase (CaMK) signaling.^1–3) DAG and IP₃ are generated from phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) by phospholipase C (PLC). Therefore, to uncover the regulatory mechanisms of generation and metabolism of PI(4,5)P₂ is important for understanding a broad range of cellular processes. Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) acts as a key enzyme to generate PI(4,5)P₂ by phosphorylating the five position on the inositol ring of phosphatidylinositol 4-phosphate (PI(4)P). It was reported that PI4P5K regulates Ca²⁺ signaling, cytoskeletal dynamics, and membrane trafficking,^4–7) and is related to Alzheimer’s disease, pain regulation, and cancer cell proliferation,^8–11) thus elucidation of the regulatory mechanisms of PI4P5K is important biologically and pharmacologically.

Previous studies reported that the essential gene its3⁺ encodes a functional homologue of mammalian PI4P5K in fission yeast.^12,13) Fission yeast Its3 converts PI(4)P to PI(4,5)P₂ and regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking. its3-1 mutant cells displayed phenotypes such as high-temperature sensitivity (TS), FK506 sensitivity (FKs), and low PI(4,5)P₂ levels.¹²⁾ To reveal the regulatory mechanisms of PI4P5K signaling, we performed a genetic screening and identified multicopy suppressors of its3-1 cells. We successfully identified puf3⁺ and puf4⁺, encoding Pumilio and FBF (PUF) family RNA-binding proteins, that when overexpressed suppressed the TS and the FKs of its3-1 cells. The PUF family of RNA-binding proteins regulates mRNA stability and translation by directly binding to the 3′untranslated region (3′UTR) of target mRNAs and is highly conserved in organisms as diverse as plants, yeast, and humans. It is reported that the PUF family proteins regulate diverse biological processes including cell cycle, differentiation, cell death, immune response, and memory.^14–21) Moreover, the PUF family proteins are associated with various diseases, including cancer, epilepsy, and infertility.^18,19,22) PUF family proteins contain a highly conserved RNA-binding domain, which is known as the Pumilio homology domain (PUM-HD) that mostly consists of eight imperfect tandem repeats of approx. 36 amino acids.²³⁾ There are nine PUF family genes puf1⁺, puf2⁺, puf3⁺, puf4⁺, puf5⁺, puf6⁺, nop9⁺, mpf1⁺ and mpf2⁺ in Schizosaccharomyces pombe (S. pombe). Here, we showed that Puf3 and Puf4 are novel key regulators of PI4P5K expression in fission yeast. Notably, while the overexpression of the cytoplasmic PUF family genes puf3⁺ and puf4⁺ suppressed the its3-1 phenotypes, the suppression by other PUF family genes except for a weak suppression by the mitotic PUF family gene mpf1⁺, was almost negligible. Interestingly, the overexpression of Puf3 and Puf4 increased the mRNA and protein expression levels of WT Its3 and mutant Its3. Our findings implicate a novel mechanism of the PI4P5K signaling regulation via the IP4P5K mRNA expression levels by mRNA-binding protein PUFs.

MATERIALS AND METHODS

Strains and Media

S. pombe strains used in this study are listed in Table 1. The complete medium (yeast extract with supplements (YES)) and the minimal medium (Edinburgh minimal medium (EMM)) have been described previously.²⁴⁾

Table 1. Schizosaccharomyces pombe Strains Used in This Study

Strain	Genotype	Source or reference
HM123	h− leu1-32	Our stock
KP167	h+ leu1-32 its3-1	Zhang et al.12)
KP857	h− leu1-32 ura4-D18 puf4::ura4+	This study
KP2282	h− leu1-32 ura4-D18 puf3::kanMX6 puf4::ura4+	This study
KP2336	h− leu1-32 puf3::kanMX6	This study
KP2573	h− leu1-32 its3-1 ura4-D18 puf4::ura4+	This study
KP2574	h− leu1-32 its3-1 puf3::kanMX6	This study
KP2575	h− leu1-32 ura4-D18 its3-1 puf3::kanMX6 puf4::ura4+	This study
SP2863	h− leu1-32 its3-GFP::kanMX6	This study
SP2879	h− leu1-32 its3-1-GFP::kanMX6	This study

Growth Conditions

Unless otherwise stated, cells were cultivated at 27 °C in EMM, or YES. Prior to several experiments, the cells were grown to the mid-log phase (OD_660 nm = 0.6).

Gene Expression

For gene expression in yeast, the thiamine-repressible nmt1 promoter was used.²⁵⁾ Expression was repressed by the addition of 4.0 µg/mL thiamine to EMM and was induced by incubating the cells in EMM lacking thiamine. The green fluorescent protein (GFP)-fused gene was subcloned into the pREP1 vector. Standard genetic and recombinant DNA methods²⁴⁾ were used except where noted.

Microscopy and Miscellaneous Methods

Light microscopy methods, such as differential interference contrast (DIC) and fluorescence microscopy, were performed as described.²⁶⁾

Protein Extraction (Alkaline Extraction) and Detection

Cell extract preparation was performed as previously described.²⁷⁾ The following primary antibodies were used: anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, U.S.A.), and anti-GFP (derived from rabbit serum).²⁸⁾ As secondary antibodies, goat horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) antibodies (Cell Signaling Technology, Danvers, MA, U.S.A.) were used. As a secondary antibody, anti-rabbit (#7074) IgG HRP-linked antibody (Cell signaling Technology) or anti-mouse (#7076) IgG HRP-linked antibody (Cell signaling Technology) were used. The proteins were detected by Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan). Relative intensities of all bands were quantified using MULTI GAUGE Ver. 3.2 software (FUJIFILM, Tokyo, Japan).

Total RNA Extraction from Fission Yeast

Cells were grown to the mid-log phase (OD660 nm = 0.5) in EMM containing thiamine. Cells were harvested by centrifugation and washed with cold water. Total cellular RNA was prepared using the hot-phenol method.²⁹⁾

Quantitative Real-Time PCR (qRT-PCR)

qRT-PCR analysis was performed using LightCycler^® 480 System (Roche, Basel, Switzerland) with LightCycler^® 480 SYBR Green I Master (Roche) using the following amplification protocol: 5 min at 95 °C followed by 45 cycles of 10 s at 95 °C, 10 s at 60 °C, and 10 s at 72 °C. Data were normalized by the Ura4 mRNA expression in each sample. Primer sets are listed in Table 2.

Table 2. List of Primers for qRT-PCR

Gene	Sequence
its3+ qRT-PCR forward	5′-CCTCCAACCCTGTAACCTCA-3′
its3+ qRT-PCR reverse	5′-AGGAGCTGAGCGTGGAAC-3′
ura4+ qRT-PCR forward	5′-GCTACAATATGCATCTGGTGTGTA-3′
ura4+ qRT-PCR reverse	5′-GCACTGTATGGCAATTTGTGA-3′

Statistical Analysis

Repeated ANOVA was used for multiple comparisons. p-Values of less than 0.05 are judged statistically significant. Values are shown as means ± standard error of the mean (S.E.M.).

RESULTS

Isolation of the Fission Yeast PUF Family Genes puf3⁺ and puf4⁺ as Multicopy Suppressors of its3-1 Cells

We performed a genetic screening utilizing the TS of its3-1 to identify genes that are functionally related to PI4P5K signaling, and isolated PUF family genes puf3⁺ and puf4⁺ as multicopy suppressors of its3-1. its3-1 cells grew at the permissive temperature of 27 °C, but the cells failed to grow at the restrictive temperature of 34 °C in EMM containing thiamine (suppressed condition) (Fig. 1A; its3-1 +vector). As shown in Fig. 1A, its3-1 cells overexpressing either puf3⁺ or puf4⁺ gene suppressed the TS of its3-1 cells at 34 °C in EMM containing thiamine. However, its3-1 cells overexpressing either puf3⁺ or puf4⁺ gene failed to grow at 36 °C in EMM containing thiamine, indicating that Puf3 and Puf4 partially suppress the TS of its3-1 cells. There are nine PUF family proteins in S. pombe³⁰⁾ (Fig. 1B), we investigated whether only Puf3 and Puf4 could suppress the TS of its3-1, or whether the other PUF family protein(s) could also suppress the TS of its3-1. Therefore, we examined the effects of the overexpression of all nine PUF family genes on the TS of its3-1. its3-1 cells exhibited TS as the cells’ growth was suppressed at 34 °C in EMM containing thiamine, whereas wild-type (WT) cells grew normally (Fig. 1A). The TS of its3-1 was partially repressed by expressing the plasmid harboring either puf3⁺ or puf4⁺ in EMM containing thiamine at 34 °C (Fig. 1A). The repression activity of puf3⁺ against the TS of its3-1 was stronger than that of puf4⁺ (Fig. 1A). Furthermore, the overexpression of mpf1⁺ also partially repressed the TS of its3-1 in EMM containing thiamine at 34 °C, but the repression activity was weaker than that of puf3⁺ and puf4⁺ (Fig. 1A). On the other hand, the overexpression of puf1⁺, puf2⁺, and puf5⁺ exacerbated the TS of its3-1 in EMM containing thiamine at 34 °C (Fig. 1A). The overexpression of the other PUF family genes puf6⁺, mpf2⁺, and nop9⁺ did not affect the TS of its3-1 in EMM containing thiamine at 34 °C (Fig. 1A). Similarly, the TS of its3-1 was partially repressed by the overexpression of puf3⁺, puf4⁺, and mpf1⁺ in YES complete medium at 31 °C, but there was no difference in the repression activity of the TS when each gene was overexpressed in its3-1 cells (Fig. 1A). It has been reported that its3-1 cells display FKs, thus it was verified whether the overexpression of PUF family gene(s) could suppress the FKs of its3-1.¹²⁾ The FKs of its3-1 was partially repressed by expressing the plasmid harboring puf3⁺, puf4⁺, or mpf1⁺ in EMM containing thiamine at 27 °C (Fig. 1A). The repression activity of puf4⁺ against the FKs of its3-1 was slightly weaker than that of puf3⁺ and mpf1⁺ (Fig. 1A). On the other hand, the overexpression of puf2⁺ exacerbated the FKs of its3-1, and the overexpression of the other PUF family genes puf1⁺, puf5⁺, puf6⁺, mpf2⁺, and nop9⁺ did not affect the FKs of its3-1 in EMM containing thiamine at 27 °C (Fig. 1A). Similarly, the FKs of its3-1 was partially repressed by the overexpression of puf3⁺, puf4⁺, and mpf1⁺ in YES complete medium at 27 °C, and the repression activities of puf3⁺ and puf4⁺ against the FKs of its3-1 were weaker than that of mpf1⁺ (Fig. 1A). The overexpression of the other PUF family genes puf1⁺, puf2⁺, puf5⁺, puf6⁺, mpf2⁺, and nop9⁺ did not affect the FKs of its3-1 in YES complete medium at 27 °C (Fig. 1A). We also examined the effects of the overexpression of all nine PUF family genes in its3-1 using EMM without thiamine (highly overexpressed condition). Importantly, the overproduction of puf1⁺, puf2⁺puf3⁺, puf4⁺, mpf1⁺, and mpf2⁺ displayed strong toxicity and that of puf6⁺ displayed weak toxicity in the media without thiamine (Fig. 1A). We decided that using EMM without thiamine was not appropriate to verify the effect of the overproduction of PUF family genes on its3-1 phenotypes.

Fig. 1. Effects of Fission Yeast Pumilios on the Phenotypes of its3-1

(A) Fission yeast Puf3 and Puf4 are the multicopy suppressors of its3-1 cells. Cells were transformed with control pREP1-GFP vector or pREP1-GFP vector containing the indicated genes and spotted onto EMM without thiamine (highly expressed condition), EMM containing thiamine (repressed condition) medium, or YES medium with/without 0.2 µg/mL FK506, then incubated at the indicated temperature. (B) Schematic diagram of fission yeast Pumilios. PUF repeat positions determined by SMART (Simple Modular Architecture Research Tool) are indicated by black circles. Amino acid (aa) positions are indicated. (C) The intracellular localization of fission yeast Pumilio proteins in its3-1 cells. The localization of GFP-fused Pumilio proteins expressed in living its3-1 cells, grown at 27 °C in an EMM medium containing thiamine (repressed condition) was analyzed by fluorescence micrography (green). The number in the picture indicates the exposure time on a fluorescence microscope. DIC, differential interference contrast image. Bar, 10 µm. (D) The expression levels of fission yeast Pumilio proteins in its3-1 cells. Western blot analysis to confirm the GFP-fused fission yeast Pumilio protein expression levels in its3-1 cells at the same condition as Fig. 1C. Tubulin was used as a reference protein. (E) The temperature sensitivity of its3-1 was exacerbated by the deletion of puf4⁺ but not puf3⁺ genes. Cells were spotted onto EMM containing Leucine medium or YES medium with/without 0.2 µg/mL FK506, then incubated at the indicated temperature. (F) The intracellular localization of the endogenous Its3-GFP or Its3-1-GFP in living cells grown at 27 °C in YES medium was analyzed by fluorescence micrography. Bar, 10 µm. (G) The intracellular localization of the endogenous Its3-1-GFP in living cells having pREP1-GST vector or pREP1-GST vector containing the indicated genes grown at 27 °C in YES medium was analyzed by fluorescence micrography. Bar, 10 µm.

Next, the intracellular localization of PUF proteins in its3-1 cells was investigated to obtain clues to the suppression mechanism of its3-1 phenotypes by Puf3 and Puf4. Puf1, Puf2, Puf3, Puf4, and meiotic Mpf2 localized to the cytoplasm, and Puf6 and nop9 localized to the nucleus in its3-1 cells (Fig. 1C). These localizations are essentially similar to the previous report on the PUF family RNA-binding protein localization in the WT cells.³¹⁾ Furthermore, Puf5 and Meiotic PUF protein Mpf1 localized to the cytoplasm and unknown cytoplasmic dot-like structures (Fig. 1C). These fluorescence microscopy results suggested that Puf3 and Puf4 act in the cytoplasm to suppress the phenotype of its3-1 cells. Further, we confirmed the protein expression levels of PUF proteins in its3-1 cells. PUF proteins other than Puf3 and Puf4 proteins were sufficiently expressed to the extent comparable to those of Puf3 and Puf4 proteins (Fig. 1D).

To reveal the detailed genetic interaction between its3⁺ and PUF family genes puf3⁺ and puf4⁺, we examined the effect of the gene knockout of puf3⁺ and/or puf4⁺ on the phenotypes of its3-1. It has been reported that the gene knockout cells of either puf3⁺ or puf4⁺ were viable in the vegetative condition, we examined whether the gene knockout of puf3⁺ and/or puf4⁺ affect the cell growth. As a result, the single gene knockout cells (Δpuf3 and Δpuf4) and the double gene knockout cells (Δpuf3 Δpuf4) grew at both 27 and 31 °C in EMM medium and at both 27 and 31 °C in YES medium similarly to the WT cells, and did not display FKs in EMM and YES medium (Fig. 1E). Moreover, the gene knockout of Puf3 did not affect the TS of its3-1, but the gene knockout of Puf4 exacerbated the TS of its3-1 (Fig. 1E). In addition, the effect of the double gene knockout of Puf3 and Puf4 on the TS of its3-1 was similar to that of the single gene knockout of Puf4 (Fig. 1E). On the other hand, single gene knock out of puf3⁺ or puf4⁺ and double gene knockout of puf3⁺ and puf4⁺ did not affect the FKs of its3-1 cells, but it is likely that the FKs of its3-1 cells were too strong to evaluate the effect of puf3⁺ and/or puf4⁺ knockout(s).

We next focused on the intracellular localization of Its3 and Its3-1 proteins. Fluorescence microscopy analysis showed that Its3-GFP localized to the plasma membrane and the cytoplasmic structures, whereas Its3-1-GFP localized to the cytoplasmic structures but not to the plasma membrane, thus suggesting the possibility that the inability of the Its3-1 protein to localize to the plasma membrane may lead to the defects in the function of Its3-1 as PI4P5K (Fig. 1F). One possible explanation for the suppression mechanism of its3-1 phenotype is that Puf3 and Puf4 normalize the intracellular localization of Its3-1. Contrary to expectations, the localization of Its3-1 was not affected by the overexpression of Puf3 or Puf4 (Fig. 1G). Consequently, it is considered that Puf3 and Puf4 do not control the intracellular localization of Its3-1.

PUF Domains of Puf3 and Puf4 Have a Critical Role to Suppress the its3-1 Cells’ Phenotypes

As mentioned above, PUF proteins bind to the target mRNAs through its RNA-binding domain (PUF domain) and regulate the stabilities of target mRNAs. To investigate if the PUF domain in Puf3/Puf4 plays a key role in the suppression of its3-1, two PUF domain-deleted mutants of Puf3 and Puf4 were constructed (ΔPUF1,2; ΔPUF3,4; ΔPUF5,6; ΔPUF7,8) and expressed in its3-1 cells. The overexpression of ΔPUF5,6 and ΔPUF7,8 abolished the suppression ability of Puf3 and Puf4 against TS and FKs of its3-1 (Fig. 2A). On the other hand, ΔPUF1,2 and ΔPUF3,4 partially retained the suppression ability of Puf3 and Puf4 against TS and FKs of its3-1 (Fig. 2A). The intracellular localization of GFP-fused PUF domain-deleted mutants of Puf3 and Puf4 in its3-1 cells was observed by fluorescence microscopy. All PUF domain mutants of Puf3 and Puf4 were localized to the cytoplasm similarly to the WT Puf3 and Puf4 (Fig. 2B). Thus, deletion of the PUF domain did not affect the cytoplasmic localization of Puf3 and Puf4 and the failure of the PUF domain-deleted mutants of Puf3 and Puf4 to suppress the its3-1 phenotypes may not be derived from the alterations of intracellular localization, but from the impaired RNA-binding activity. Further, the protein expression levels of PUF domain-deleted mutants of Puf3 and Puf4 in its3-1 cells were confirmed by Western blot analysis and were sufficiently expressed (Fig. 2C).

Fig. 2. PUF Domain Deletions of Puf3 and Puf4 Reduced the Activity to Suppress the Phenotypes of its3-1

(A) C-Terminus PUF domains of Puf3 and Puf4 are essential for the suppression of its3-1 phenotype. Cells were transformed with control pREP1-GFP vector or pREP1-GFP vector containing the indicated genes and spotted onto EMM containing thiamine (repressed condition) medium with/without 0.2 µg/mL FK506, then incubated at the indicated temperature. (B) The intracellular localization of the PUF domain deleted Puf3 and Puf4 proteins in its3-1 cells. The localization of GFP-fused Pumilio proteins expressed in living its3-1 cells, grown at 27 °C in an EMM medium containing thiamine (repressed condition) was analyzed by fluorescence micrography (green). Bar, 10 µm. (C) The expression levels of the PUF domain deleted Puf3 and Puf4 proteins in its3-1 cells. Western blot analysis to confirm the GFP-fused PUF domain deleted Puf3 and Puf4 protein expression levels in its3-1 cells at the same condition as Fig. 2B. Tubulin was used as a reference protein.

The Overproduction of its3-1 Gene Suppressed its3-1 Cells’ Phenotypes

It is expected that its3-1 encodes a dysfunctional PI4P5K mutant because its3⁺ is an essential gene and PI(4,5)P₂ levels are decreased in its3-1 cells at the restrictive temperature. Therefore, if Its3-1 does not completely lose its function, it is possible that the function of Its3-1 and its3-1 phenotypes can be suppressed by overexpressing not only WT Its3 but also mutant Its3-1. Thus, it was examined whether the phenotypes of its3-1 were suppressed by the overproduction of its3⁺ or its3-1. As a result, the TS, but not that of the FKs of its3-1 was not suppressed by its3⁺ overproduction in the EMM medium (Fig. 3). It should be noted that the overexpression of its3 inhibited its3-1 cells’ growth in EMM medium at the permissive temperature (Fig. 3; EMM 27 °C). The TS and the FKs of its3-1 were suppressed by its3⁺ overproduction in YES medium (Fig. 3). Importantly, overexpression of its3-1 complemented the phenotypes of its3-1 (Fig. 3). These results suggested that one of the mechanisms by which the its3-1 phenotypes is suppressed is increasing the protein expression level of Its3-1.

Fig. 3. The Overproduction of Mutated Its3 (Its3-1) Suppressed the Phenotypes of its3-1

Cells were transformed with control pREP1-GFP vector or pREP1-GFP vector containing the indicated genes and spotted onto EMM containing thiamine (repressed condition) medium or YES medium with/without 0.2 µg/mL FK506, then incubated at the indicated temperature.

Puf3 and Puf4 Increased the Protein and mRNA Expression Levels of Its3

The above results raise the possibility that Puf3 and Puf4 when overproduced may suppress the phenotypes of its3-1 by regulating the Its3 protein expression levels. Therefore, we investigated the effect of the overproduction of Puf3 and Puf4 on Its3 and Its3-1 protein expression levels. Interestingly, the protein expression level of Its3-1-GFP was lower than that of Its3-GFP (Fig. 4A). It was also shown that the protein expression level of Its3-GFP increased about 3-fold by Puf3 overproduction and about 4-fold by Puf4 overproduction (Fig. 4A). Furthermore, it was shown that the protein expression level of Its3-1-GFP was increased about 2-fold due to overexpression of Puf3 and Puf4 (Fig. 4A).

Fig. 4. The Overproduction of Puf3 and Puf4 Increased the mRNA and Protein Expression Levels of Its3 and Its3-1

(A) Overproduction of Puf3 and Puf4 induced Its3 and Its3-1 protein levels. Western blot analysis to evaluate the relative Its3 or Its3-1 protein expression levels in the cells having pREP1-GST vector or pREP1-GST vector containing the indicated genes grown at 27 °C in EMM containing thiamine (repressed condition) medium. Tubulin was used as a reference protein. Relative Its3 protein expression levels in the cells having pREP1-GST vector were set as 1. The data were averaged from seven independent experiments (n = 3). Columns, mean; bars, S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.005, n.s., not significant; significantly different from pREP1-GST vector in each cell using one-way ANOVA, followed by a post hoc test using Dunnett’s multiple comparisons. Comparisons between Its3-GFP +vector cells and Its3-1-GFP +vector cells were made by one-way ANOVA, followed by a post hoc test using Dunnett’s multiple comparisons. (B) qRT-PCR analysis to evaluate the relative Its3 or Its3-1 mRNA expression levels in the cells having pREP1-GST vector or pREP1-GST vector containing the indicated genes grown at 27 °C in EMM containing thiamine (repressed condition) medium. ura4⁺ was used as a reference gene. Relative Its3 mRNA expression levels in the cells having pREP1-GST vector were set as 1. The data were averaged from seven independent experiments (n = 20). Columns, means; bars, SEM. n.s., not significant; significantly different from pREP1-GST vector in each cell using one-way ANOVA, followed by a post hoc test using Dunnett’s multiple comparisons. Comparisons between Its3-GFP +vector cells and Its3-1-GFP +vector cells were made by one-way ANOVA, followed by a post hoc test using Dunnett’s multiple comparisons.

Next, we focused on Its3 mRNA expression levels, because Puf3 and Puf4 are PUF family mRNA-binding proteins that posttranscriptionally regulate their target mRNAs. Therefore, relative Its3 and Its3-1 mRNA expression levels were detected by qRT-PCR under the same conditions used for verifying Its3 and Its3-1 protein expression levels in Fig. 4A. Consistent with the results obtained with the Its3 and Its3-1 protein expression levels, the mRNA expression level of Its3-1-GFP was also lower than that of Its3-GFP (Fig. 4B). Moreover, the mRNA expression levels of both Its3-GFP and Its3-1-GFP also significantly increased by overexpression of Puf3 and Puf4. These results strongly suggest that Puf3 and Puf4 function to increase the expression level of Its3 mRNA, which leads to increased expression levels of Its3 protein. However, these results do not exclude the possibility that Puf3 and Puf4 regulate the translation efficiency of Its3 mRNA or proteolytic degradation of Its3 protein.

DISCUSSION

In this study, we identified PUF family proteins Puf3 and Puf4 as novel regulators of PI4P5K, which is an important signaling factor in the phosphatidylinositol metabolism associated with several biological processes and diseases such as neurodegenerative disease and cancer. Overproduction of Puf3 or Puf4 suppressed the phenotypes of its3-1 cells and significantly increase the expression levels of Its3 and Its3-1 proteins and mRNAs. These results indicate that the overexpression of Puf3 and Puf4 recovers the decreased expression level of PI(4,5)P₂ and increases the production of IP₃ and DAG by increasing the Its3-1 protein expression level in its3-1 cells. Our findings propose a new mechanism by which PUF proteins Puf3 and Puf4 increase the protein expression levels of PI4P5K presumably through the posttranscriptional regulation of PI4P5K mRNA to regulate the phosphatidylinositol metabolism. However, our data do not rule out the possibility that Puf3 and Puf4 suppress the phenotypes of its3-1 cells through the other PI kinases. PI(4)P is accumulated, and PI(4,5)P₂ is decreased in its3-1 cells at the restrictive temperature.¹²⁾ In addition, Phospholipase C Plc1 has also been identified as another multicopy suppressor of its3-1 cells, Plc1 hydrolyzed PI(4,5)P₂ and provided IP₃ and DAG that was decreasing in its3-1 cells. Therefore, despite the decreased production of PI(4,5)P₂ in its3-1 cells, overexpression of Plc1, which degrades PI(4,5)P₂ and produces IP₃ and DAG, suppresses the phenotypes of its3-1 cells. These results suggest that the production of PI(4,5)P₂ in its3-1 cells is not related to the its3-1 phenotypes, but the increased production of IP₃ and DAG is crucial for the suppression of its3-1 phenotypes. Moreover, these previous reports raise the possibility that Puf3 and Puf4 suppress the its3-1 phenotypes through factors involved in the synthesis of PI(4)P, PI(4,5)P₂, IP_3, and DAG.

As a result of investigating the effects of overproductions of all nine PUF family genes in S. pombe on its3-1 phenotypes, mitotic PUF family gene mpf1⁺ also suppressed its3-1 phenotypes in addition to puf3⁺ and puf4⁺ (Fig. 1B). There have been no reports on the function of Mpf1 so far, but interestingly, Mpf1 was localized in the cytoplasm and unknown cytoplasmic dot-like structures in its3-1 cells in nutrient-rich medium (Fig. 1C). These results were shown by using exogenously expressed GFP-Mpf1 by the nmt1 promoter, thus it is unclear whether the endogenous Mpf1 is also expressed in a mitotic state and shows similar localization. However, it should be considered that Mpf1 may have some physiological function even in the mitotic state. Importantly, its3-1 phenotypes were exacerbated by Puf4 knockout, but not by Puf3 knockout, suggesting that Puf4 has unique functions for its3-1 and there is no factor that replaces the functions of Puf4, while the function of Puf3 for its3-1 can be replaced by other factors. Mpf1 is one of the candidates that have alternative functions of Puf3.

A recent study showed that the fission yeast Puf2 and Puf4 regulate iron uptake through frp1⁺ encoding a key enzyme of the reductive iron uptake pathway.³²⁾ Puf2 and Puf4 may protect cells against toxic levels of iron by binding to the Pumilio response elements (PREs) in the 3′UTR of Frp1 mRNA under basal and iron-replete conditions, and destabilizing Frp1 mRNA. Notably, Its3 mRNA contains a potential canonical PRE sequence (UGUANAUA) in its 3′UTR,^33,34) indicating that Puf3 and Puf4 might be able to directly bind to and regulate Its3 mRNA. Furthermore, the PUF domain deleted mutants of Puf3 and Puf4 decreased the ability to suppress its3-1 phenotypes, indicating that Puf3 and Puf4 suppress its3-1 phenotypes through their RNA-binding activity. Combined with the results of Puf3 or Puf4 overproduction that increased the expression levels of both Its3 mRNA and Its3 protein levels, it is hypothesized that Puf3 and Puf4 recognize and bind to the PRE sequence in the 3′UTR of Its3 mRNA and post-transcriptionally regulate Its3 expression levels. It is known that PI4P5K is a potential target for cancers with KRAS or TP53 mutations.³⁵⁾ Our findings suggest the existence of a novel mechanism to regulate PI4P5K expression levels by RNA-binding protein PUFs, which we believe to be the bases for drug development targeting the expression of PI4P5K.

Acknowledgments

We would like to thank William Figoni for his critical reading of the manuscript. This research was funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities, 2014–2018 [JPS1411037 (R. Sugiura)], a Grant-in-Aid for scientific research from the Japan Society for the Promotion of Science (JSPS) KAKENHI [JP19H03376 (R. Sugiura), JP20K06494 (R. Satoh)], Faculty Assistance and Development Research Grant of Kindai University Research Enhancement Grant [SR04, 2021 (R. Satoh)] and a Grant by the Antiaging Project for Private Universities (R. Sugiura).

Conflict of Interest

The authors declare no conflict of interest.

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