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Rpm2p, a protein subunit of mitochondrial RNase P, physically and genetically interacts with cytoplasmic processing bodies.

Stribinskis V, Ramos KS - Nucleic Acids Res. (2007)

Bottom Line: When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide.The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin.Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Center for Genetics and Molecular Medicine, School of Medicine, University of Louisville, Louisville, KY, USA. v0stri01@louisville.edu

ABSTRACT
The RPM2 gene of Saccharomyces cerevisiae codes for a protein subunit of mitochondrial RNase P and has another unknown essential function. We previously demonstrated that Rpm2p localizes to the nucleus and acts as a transcriptional activator. Rpm2p influences the level of mRNAs that encode components of the mitochondrial import apparatus and essential mitochondrial chaperones. Evidence is presented here that Rpm2p interacts with Dcp2p, a subunit of mRNA decapping enzyme in the two-hybrid assay, and is enriched in cytoplasmic P bodies, the sites of mRNA degradation and storage in yeast and mammalian cells. When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide. Proteasome mutants, ump1-2 and pre4-2, that bypass essential Rpm2p function, also stabilize P bodies. The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin. Genetic analysis revealed that overexpression of Dhh1p (a DEAD box helicase localized to P bodies) suppresses temperature-sensitive growth of the rpm2-100 mutant. Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism. The results presented here, and the transcriptional activation function demonstrated earlier, implicate Rpm2p as a coordinator of transcription and mRNA storage/decay in P bodies.

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Growth of rpm2-100 strains. The rpm2-100 transformants carrying either RPM2, DHH1 or PAB1 genes under expression of GAL1 promoter or vector alone were plated on synthetic plates containing galactose and scored for growth at 30 and 37°C.
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Figure 1: Growth of rpm2-100 strains. The rpm2-100 transformants carrying either RPM2, DHH1 or PAB1 genes under expression of GAL1 promoter or vector alone were plated on synthetic plates containing galactose and scored for growth at 30 and 37°C.

Mentions: Previous studies have showed that the rpm2-100 mutation causes loss of cell osmotic integrity at the non-permissive temperature, and this phenotype can be suppressed by increasing osmolarity of the growth medium (Stribinskis et al., manuscript in preparation). We have used this temperature-sensitive growth phenotype to isolate high-copy suppressors that allow rpm2-100 mutant cells to grow at the restrictive temperature. The yeast S. cerevisiae cDNA library under control of the GAL1-regulated promoter (14) was introduced into rpm2-100 cells; transformants were plated on synthetic plates containing galactose as the sole carbon source and incubated at 37°C. The screen revealed that in addition to Rpm2p, Dhh1p, an RNA helicase (16) and Pab1p, poly (A)-binding protein (17,18), are high-copy suppressors of rpm2-100 temperature-sensitive growth (Figure 1). Interestingly, PAB1 was found as a high-copy suppressor of an rpm2 deletion strain in an independent genetic screen (Nancy C. Martin, personal communication). Pab1 is a multifunctional protein that plays a role in stabilization of mRNAs, brings the 5′ and 3′ ends of mRNAs into proximity by binding eIF4G, and stimulates translation (19–21). In addition, recent observations demonstrate a role for Pab1p in mRNA export from the nucleus (22,23).Figure 1.


Rpm2p, a protein subunit of mitochondrial RNase P, physically and genetically interacts with cytoplasmic processing bodies.

Stribinskis V, Ramos KS - Nucleic Acids Res. (2007)

Growth of rpm2-100 strains. The rpm2-100 transformants carrying either RPM2, DHH1 or PAB1 genes under expression of GAL1 promoter or vector alone were plated on synthetic plates containing galactose and scored for growth at 30 and 37°C.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851656&req=5

Figure 1: Growth of rpm2-100 strains. The rpm2-100 transformants carrying either RPM2, DHH1 or PAB1 genes under expression of GAL1 promoter or vector alone were plated on synthetic plates containing galactose and scored for growth at 30 and 37°C.
Mentions: Previous studies have showed that the rpm2-100 mutation causes loss of cell osmotic integrity at the non-permissive temperature, and this phenotype can be suppressed by increasing osmolarity of the growth medium (Stribinskis et al., manuscript in preparation). We have used this temperature-sensitive growth phenotype to isolate high-copy suppressors that allow rpm2-100 mutant cells to grow at the restrictive temperature. The yeast S. cerevisiae cDNA library under control of the GAL1-regulated promoter (14) was introduced into rpm2-100 cells; transformants were plated on synthetic plates containing galactose as the sole carbon source and incubated at 37°C. The screen revealed that in addition to Rpm2p, Dhh1p, an RNA helicase (16) and Pab1p, poly (A)-binding protein (17,18), are high-copy suppressors of rpm2-100 temperature-sensitive growth (Figure 1). Interestingly, PAB1 was found as a high-copy suppressor of an rpm2 deletion strain in an independent genetic screen (Nancy C. Martin, personal communication). Pab1 is a multifunctional protein that plays a role in stabilization of mRNAs, brings the 5′ and 3′ ends of mRNAs into proximity by binding eIF4G, and stimulates translation (19–21). In addition, recent observations demonstrate a role for Pab1p in mRNA export from the nucleus (22,23).Figure 1.

Bottom Line: When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide.The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin.Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Center for Genetics and Molecular Medicine, School of Medicine, University of Louisville, Louisville, KY, USA. v0stri01@louisville.edu

ABSTRACT
The RPM2 gene of Saccharomyces cerevisiae codes for a protein subunit of mitochondrial RNase P and has another unknown essential function. We previously demonstrated that Rpm2p localizes to the nucleus and acts as a transcriptional activator. Rpm2p influences the level of mRNAs that encode components of the mitochondrial import apparatus and essential mitochondrial chaperones. Evidence is presented here that Rpm2p interacts with Dcp2p, a subunit of mRNA decapping enzyme in the two-hybrid assay, and is enriched in cytoplasmic P bodies, the sites of mRNA degradation and storage in yeast and mammalian cells. When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide. Proteasome mutants, ump1-2 and pre4-2, that bypass essential Rpm2p function, also stabilize P bodies. The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin. Genetic analysis revealed that overexpression of Dhh1p (a DEAD box helicase localized to P bodies) suppresses temperature-sensitive growth of the rpm2-100 mutant. Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism. The results presented here, and the transcriptional activation function demonstrated earlier, implicate Rpm2p as a coordinator of transcription and mRNA storage/decay in P bodies.

Show MeSH
Related in: MedlinePlus