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Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

Chatenay-Lapointe M, Shadel GS - PLoS ONE (2011)

Bottom Line: Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility.Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling.Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

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Over-expression of Slf1p increases respiration and CLS, and expression of Sls1p is repressed by Puf3p.(A) SLF1 DNA sequence depicting the stop codon (bold) and Puf3 element (underlined). (B) Mitochondrial oxygen consumption of wild-type cells carrying an empty vector (YEp351) or YEp351 with SLF1 during early stationary phase. (C) Western blot analysis of protein extracts derived from the same strains in (B) (representative of three biological replicates). (D) CLS analysis of the same strains in (B). (E) Q-RT-PCR analysis COX17, COX12, and RIP1 transcript levels at early stationary phase from the same strains in (B). Analysis of Slf1p levels by western blot (F) and SLF1 mRNA levels by RT-PCR (G) in wild-type and puf3Δ strains in log-phase growth (representative of three biological replicates).
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pone-0020441-g007: Over-expression of Slf1p increases respiration and CLS, and expression of Sls1p is repressed by Puf3p.(A) SLF1 DNA sequence depicting the stop codon (bold) and Puf3 element (underlined). (B) Mitochondrial oxygen consumption of wild-type cells carrying an empty vector (YEp351) or YEp351 with SLF1 during early stationary phase. (C) Western blot analysis of protein extracts derived from the same strains in (B) (representative of three biological replicates). (D) CLS analysis of the same strains in (B). (E) Q-RT-PCR analysis COX17, COX12, and RIP1 transcript levels at early stationary phase from the same strains in (B). Analysis of Slf1p levels by western blot (F) and SLF1 mRNA levels by RT-PCR (G) in wild-type and puf3Δ strains in log-phase growth (representative of three biological replicates).

Mentions: The vast majority of genes in the Puf3 regulon are regulators of mitochondrial translation (e.g. mitochondrial ribosomal proteins and translation factors) or processes immediately downstream (e.g. OXPHOS assembly factors) that control respiration. To find additional genes potentially involved in mitochondrial function, others identified transcripts that were both localized to the mitochondrial periphery and clustered with P3E mRNAs as a function of the yeast metabolic cycle (YMC) [22]. We hypothesized that a subset of the Puf3 regulon is not involved with mitochondrial translation directly, but may nonetheless be important for mitochondrial respiration. To test this hypothesis, we queried the YMC microarray database SCEPTRANS [30] for mRNAs that displayed a YMC pattern of expression, but are not predicted to localize to mitochondria. Of the top 50 transcripts coregulated with COX17 in the YMC, three met this criterion: YOR248W, a dubious ORF; SRL1, a mannoprotein associated with the cell wall [31]; and SLF1, and RNA-binding protein associated with cytoplasmic polysomes [32]. A consensus P3E exists shortly after the stop codon in the SLF1 mRNA (Figure 7A), consistent with it being a target of Puf3p. Thus Slf1p seemed like a likely candidate factor that could potentially regulate respiration and was studied further. While slf1Δ strains did not reveal any obvious growth or respiration phenotype in fermentable or non-fermentable media, over-expression of Slf1p resulted in a modest, but reproducible increase in mitochondrial oxygen consumption (Figure 7B), a pronounced increase in Cox4p, and modest increases in Cox2p and Pet100p (Figure 7C). Over-expression of Slf1p also significantly increased median CLS (Figure 7D) and significantly reduced the amount of the Puf3-target transcript, COX17, while having minimal or no effect on non-puf3-target transcripts (Figure 7E). Finally, Slf1p protein (Figure 7F), but not its transcript (Figure 7G), was up-regulated in a puf3Δ strain. Altogether, these data indicate that Slf1p a P3E-containing gene that encodes a putative regulator of mitochondrial function and CLS and is repressed by Puf3p at the post-transcriptional level.


Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

Chatenay-Lapointe M, Shadel GS - PLoS ONE (2011)

Over-expression of Slf1p increases respiration and CLS, and expression of Sls1p is repressed by Puf3p.(A) SLF1 DNA sequence depicting the stop codon (bold) and Puf3 element (underlined). (B) Mitochondrial oxygen consumption of wild-type cells carrying an empty vector (YEp351) or YEp351 with SLF1 during early stationary phase. (C) Western blot analysis of protein extracts derived from the same strains in (B) (representative of three biological replicates). (D) CLS analysis of the same strains in (B). (E) Q-RT-PCR analysis COX17, COX12, and RIP1 transcript levels at early stationary phase from the same strains in (B). Analysis of Slf1p levels by western blot (F) and SLF1 mRNA levels by RT-PCR (G) in wild-type and puf3Δ strains in log-phase growth (representative of three biological replicates).
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pone-0020441-g007: Over-expression of Slf1p increases respiration and CLS, and expression of Sls1p is repressed by Puf3p.(A) SLF1 DNA sequence depicting the stop codon (bold) and Puf3 element (underlined). (B) Mitochondrial oxygen consumption of wild-type cells carrying an empty vector (YEp351) or YEp351 with SLF1 during early stationary phase. (C) Western blot analysis of protein extracts derived from the same strains in (B) (representative of three biological replicates). (D) CLS analysis of the same strains in (B). (E) Q-RT-PCR analysis COX17, COX12, and RIP1 transcript levels at early stationary phase from the same strains in (B). Analysis of Slf1p levels by western blot (F) and SLF1 mRNA levels by RT-PCR (G) in wild-type and puf3Δ strains in log-phase growth (representative of three biological replicates).
Mentions: The vast majority of genes in the Puf3 regulon are regulators of mitochondrial translation (e.g. mitochondrial ribosomal proteins and translation factors) or processes immediately downstream (e.g. OXPHOS assembly factors) that control respiration. To find additional genes potentially involved in mitochondrial function, others identified transcripts that were both localized to the mitochondrial periphery and clustered with P3E mRNAs as a function of the yeast metabolic cycle (YMC) [22]. We hypothesized that a subset of the Puf3 regulon is not involved with mitochondrial translation directly, but may nonetheless be important for mitochondrial respiration. To test this hypothesis, we queried the YMC microarray database SCEPTRANS [30] for mRNAs that displayed a YMC pattern of expression, but are not predicted to localize to mitochondria. Of the top 50 transcripts coregulated with COX17 in the YMC, three met this criterion: YOR248W, a dubious ORF; SRL1, a mannoprotein associated with the cell wall [31]; and SLF1, and RNA-binding protein associated with cytoplasmic polysomes [32]. A consensus P3E exists shortly after the stop codon in the SLF1 mRNA (Figure 7A), consistent with it being a target of Puf3p. Thus Slf1p seemed like a likely candidate factor that could potentially regulate respiration and was studied further. While slf1Δ strains did not reveal any obvious growth or respiration phenotype in fermentable or non-fermentable media, over-expression of Slf1p resulted in a modest, but reproducible increase in mitochondrial oxygen consumption (Figure 7B), a pronounced increase in Cox4p, and modest increases in Cox2p and Pet100p (Figure 7C). Over-expression of Slf1p also significantly increased median CLS (Figure 7D) and significantly reduced the amount of the Puf3-target transcript, COX17, while having minimal or no effect on non-puf3-target transcripts (Figure 7E). Finally, Slf1p protein (Figure 7F), but not its transcript (Figure 7G), was up-regulated in a puf3Δ strain. Altogether, these data indicate that Slf1p a P3E-containing gene that encodes a putative regulator of mitochondrial function and CLS and is repressed by Puf3p at the post-transcriptional level.

Bottom Line: Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility.Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling.Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

Show MeSH
Related in: MedlinePlus