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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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The Regulation of Respiration by Puf3p is Independent of Mitochondrial Biogenesis Pathways.Shown is the analysis of wild-type (wt) and puf3Δ strains with plasmids over-expressing HAP4 and GSM1 (HAP4 GSM1) and corresponding empty vectors (vectors) (A) Mitochondrial oxygen consumption during late-log phase of growth. (B) Western blot analysis (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in late log phase of growth. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.
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pone-0020441-g002: The Regulation of Respiration by Puf3p is Independent of Mitochondrial Biogenesis Pathways.Shown is the analysis of wild-type (wt) and puf3Δ strains with plasmids over-expressing HAP4 and GSM1 (HAP4 GSM1) and corresponding empty vectors (vectors) (A) Mitochondrial oxygen consumption during late-log phase of growth. (B) Western blot analysis (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in late log phase of growth. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.

Mentions: To probe further whether up-regulation of mitochondrial respiration in puf3Δ strains was independent of mitochondrial biogenesis pathways, we over-expressed Hap4p, a known transcriptional regulator of mitochondrial biogenesis, and Gsm1p, another transcription factor implicated in regulating the expression of OXPHOS proteins [28], [29], in wild-type and puf3Δ strains. Simultaneous over-expression of these two transcription factors caused similar increases in respiration in both wild-type and puf3Δ strains (Figure 2A) and, further increases in the already heightened steady-state levels of Cox2p and Cox4p in the puf3Δ strain (Figure 2B). Finally, we assayed the steady-state levels of COX17 transcripts, a canonical Puf3 target [15], and two non-P3E-target transcripts, RIP1 and COX12, by quantitative real-time RT-PCR. As expected, deletion of PUF3 leads to a dramatic increase in COX17 transcripts, but had little or no effect on the non-P3E targets, while over-expression of HAP4 and GSM1 in a wild-type strain led to an increase in COX12 and RIP1 mRNA (Figure 2C). Importantly, Hap4p/Gsm1p over-expression increased all of these transcripts to a similar degree in the puf3Δ strain (Figure 2C), bolstering our conclusion that up-regulation of respiration in the absence of Puf3p is largely independent of pathways that increase mitochondrial biogenesis.


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)

The Regulation of Respiration by Puf3p is Independent of Mitochondrial Biogenesis Pathways.Shown is the analysis of wild-type (wt) and puf3Δ strains with plasmids over-expressing HAP4 and GSM1 (HAP4 GSM1) and corresponding empty vectors (vectors) (A) Mitochondrial oxygen consumption during late-log phase of growth. (B) Western blot analysis (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in late log phase of growth. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105058&req=5

pone-0020441-g002: The Regulation of Respiration by Puf3p is Independent of Mitochondrial Biogenesis Pathways.Shown is the analysis of wild-type (wt) and puf3Δ strains with plasmids over-expressing HAP4 and GSM1 (HAP4 GSM1) and corresponding empty vectors (vectors) (A) Mitochondrial oxygen consumption during late-log phase of growth. (B) Western blot analysis (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in late log phase of growth. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.
Mentions: To probe further whether up-regulation of mitochondrial respiration in puf3Δ strains was independent of mitochondrial biogenesis pathways, we over-expressed Hap4p, a known transcriptional regulator of mitochondrial biogenesis, and Gsm1p, another transcription factor implicated in regulating the expression of OXPHOS proteins [28], [29], in wild-type and puf3Δ strains. Simultaneous over-expression of these two transcription factors caused similar increases in respiration in both wild-type and puf3Δ strains (Figure 2A) and, further increases in the already heightened steady-state levels of Cox2p and Cox4p in the puf3Δ strain (Figure 2B). Finally, we assayed the steady-state levels of COX17 transcripts, a canonical Puf3 target [15], and two non-P3E-target transcripts, RIP1 and COX12, by quantitative real-time RT-PCR. As expected, deletion of PUF3 leads to a dramatic increase in COX17 transcripts, but had little or no effect on the non-P3E targets, while over-expression of HAP4 and GSM1 in a wild-type strain led to an increase in COX12 and RIP1 mRNA (Figure 2C). Importantly, Hap4p/Gsm1p over-expression increased all of these transcripts to a similar degree in the puf3Δ strain (Figure 2C), bolstering our conclusion that up-regulation of respiration in the absence of Puf3p is largely independent of pathways that increase mitochondrial biogenesis.

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