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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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Rpm2p and Dcp2p colocalize to P bodies. Cells carrying both GFP-RPM2 and DCP2-RFP in Δdcp2 strain (A); and GFP-RPM2 and LSM1-RFP in either wt or Δdcp2 strain (B), were grown in selective medium in the presence of galactose for 6 h, fixed and fluorescence determined using microscope.
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Figure 3: Rpm2p and Dcp2p colocalize to P bodies. Cells carrying both GFP-RPM2 and DCP2-RFP in Δdcp2 strain (A); and GFP-RPM2 and LSM1-RFP in either wt or Δdcp2 strain (B), were grown in selective medium in the presence of galactose for 6 h, fixed and fluorescence determined using microscope.

Mentions: We found that the essential portion of Rpm2p, which can also support mitochondrial RNase P activity under respiratory growth conditions (6), expressed as a fusion protein with GFP, concentrates in the nucleus and localizes to distinct foci in the cytoplasm that did not appear to colocalize with any organelle (8). The observation that Rpm2p interacts with Dcp2p, which resides in cytoplasmic mRNA processing bodies, P bodies (33), suggested that these foci might be P bodies. DCP2 is an essential gene, however, yeast strain yRP1358 has an unknown genetic variation that allows the Δdcp2 mutant to grow, albeit poorly at all temperatures (32). To determine whether Rpm2p colocalizes with Dcp2p, the yeast strain yRP1358 was transformed with GFP-RPM2 and DCP2-RFP constructs and their localization after induced expression of GFP-Rpm2p with galactose was determined by fluorescence microscopy. Both proteins colocalize in discrete cytoplasmic foci, and colocalization occurs in all cells that have both, green and red fluorescence (Figure 3A). In addition, overexpression of GFP-Rpm2p from a powerful, galactose-inducible promoter, neither leads to an increase in P body size nor number. The same result was also obtained in two other widely used laboratory yeast strains W303 and BY4741 (not shown). Therefore, Rpm2p, lacking a mitochondrial leader sequence, but able to support the essential function, localizes to P bodies.Figure 3.


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

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

Rpm2p and Dcp2p colocalize to P bodies. Cells carrying both GFP-RPM2 and DCP2-RFP in Δdcp2 strain (A); and GFP-RPM2 and LSM1-RFP in either wt or Δdcp2 strain (B), were grown in selective medium in the presence of galactose for 6 h, fixed and fluorescence determined using microscope.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 3: Rpm2p and Dcp2p colocalize to P bodies. Cells carrying both GFP-RPM2 and DCP2-RFP in Δdcp2 strain (A); and GFP-RPM2 and LSM1-RFP in either wt or Δdcp2 strain (B), were grown in selective medium in the presence of galactose for 6 h, fixed and fluorescence determined using microscope.
Mentions: We found that the essential portion of Rpm2p, which can also support mitochondrial RNase P activity under respiratory growth conditions (6), expressed as a fusion protein with GFP, concentrates in the nucleus and localizes to distinct foci in the cytoplasm that did not appear to colocalize with any organelle (8). The observation that Rpm2p interacts with Dcp2p, which resides in cytoplasmic mRNA processing bodies, P bodies (33), suggested that these foci might be P bodies. DCP2 is an essential gene, however, yeast strain yRP1358 has an unknown genetic variation that allows the Δdcp2 mutant to grow, albeit poorly at all temperatures (32). To determine whether Rpm2p colocalizes with Dcp2p, the yeast strain yRP1358 was transformed with GFP-RPM2 and DCP2-RFP constructs and their localization after induced expression of GFP-Rpm2p with galactose was determined by fluorescence microscopy. Both proteins colocalize in discrete cytoplasmic foci, and colocalization occurs in all cells that have both, green and red fluorescence (Figure 3A). In addition, overexpression of GFP-Rpm2p from a powerful, galactose-inducible promoter, neither leads to an increase in P body size nor number. The same result was also obtained in two other widely used laboratory yeast strains W303 and BY4741 (not shown). Therefore, Rpm2p, lacking a mitochondrial leader sequence, but able to support the essential function, localizes to P bodies.Figure 3.

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