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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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Disassembly of P bodies by translational inhibitor cycloheximide (CHX) does not occur in cells overexpressing GFP-Rpm2p. Cells expressing either Dcp2p-RFP or both, Dcp2p-RFP and GFP-Rpm2p were grown in galactose synthetic medium and P bodies visualized at different cell culture densities after incubation with or without CHX for 30 min. The different cell densities at A600 are indicated at the right side. For observation, cells were washed three times in water with or without cycloheximide without fixation.
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Figure 4: Disassembly of P bodies by translational inhibitor cycloheximide (CHX) does not occur in cells overexpressing GFP-Rpm2p. Cells expressing either Dcp2p-RFP or both, Dcp2p-RFP and GFP-Rpm2p were grown in galactose synthetic medium and P bodies visualized at different cell culture densities after incubation with or without CHX for 30 min. The different cell densities at A600 are indicated at the right side. For observation, cells were washed three times in water with or without cycloheximide without fixation.

Mentions: To determine whether association of Rpm2p with P bodies remains under conditions that promote dissociation of other known P body components, cells were exposed for 30 min to cycloheximide (100 μg/ml), an inhibitor of translation elongation. Cycloheximide induces dissociation of P bodies in both yeast and mammalian cells (33,35). Figure 4 shows that the typical localization of Dcp2p-RFP to P bodies is not observed after cycloheximide treatment. In contrast, Dcp2p-RFP remains in P bodies upon expression of GFP-Rpm2 protein in the presence of cycloheximide. Moreover, both proteins remain in P bodies under these conditions. We also examined whether the observed effect of Rpm2p on P body stability depends on the stage of growth of a yeast culture. We found that at each stage of growth, from OD600 = 0.5 (early-log phase) to OD600 = 4.0 (end of log phase), both Dcp2p and Rpm2p fluorescent-tagged fusions were present in P bodies after cycloheximide treatment (Figure 4). Therefore, overexpression of GFP-Rpm2p from an inducible GAL promoter stabilizes P bodies against dissociation by cycloheximide.Figure 4.


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

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

Disassembly of P bodies by translational inhibitor cycloheximide (CHX) does not occur in cells overexpressing GFP-Rpm2p. Cells expressing either Dcp2p-RFP or both, Dcp2p-RFP and GFP-Rpm2p were grown in galactose synthetic medium and P bodies visualized at different cell culture densities after incubation with or without CHX for 30 min. The different cell densities at A600 are indicated at the right side. For observation, cells were washed three times in water with or without cycloheximide without fixation.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 4: Disassembly of P bodies by translational inhibitor cycloheximide (CHX) does not occur in cells overexpressing GFP-Rpm2p. Cells expressing either Dcp2p-RFP or both, Dcp2p-RFP and GFP-Rpm2p were grown in galactose synthetic medium and P bodies visualized at different cell culture densities after incubation with or without CHX for 30 min. The different cell densities at A600 are indicated at the right side. For observation, cells were washed three times in water with or without cycloheximide without fixation.
Mentions: To determine whether association of Rpm2p with P bodies remains under conditions that promote dissociation of other known P body components, cells were exposed for 30 min to cycloheximide (100 μg/ml), an inhibitor of translation elongation. Cycloheximide induces dissociation of P bodies in both yeast and mammalian cells (33,35). Figure 4 shows that the typical localization of Dcp2p-RFP to P bodies is not observed after cycloheximide treatment. In contrast, Dcp2p-RFP remains in P bodies upon expression of GFP-Rpm2 protein in the presence of cycloheximide. Moreover, both proteins remain in P bodies under these conditions. We also examined whether the observed effect of Rpm2p on P body stability depends on the stage of growth of a yeast culture. We found that at each stage of growth, from OD600 = 0.5 (early-log phase) to OD600 = 4.0 (end of log phase), both Dcp2p and Rpm2p fluorescent-tagged fusions were present in P bodies after cycloheximide treatment (Figure 4). Therefore, overexpression of GFP-Rpm2p from an inducible GAL promoter stabilizes P bodies against dissociation by cycloheximide.Figure 4.

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