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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 interacts with Dcp2p in the yeast two-hybrid assay. Yeast CG1945 strains expressing the combination Gal4p BD alone or fusion, and Gal4p AD alone or fusion were spotted on SD-Trp-Leu and SD-Trp-Leu-His (in the absence or presence of aminotriazole, AT) plates and incubated until visible colonies were formed.
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Figure 2: Rpm2p interacts with Dcp2p in the yeast two-hybrid assay. Yeast CG1945 strains expressing the combination Gal4p BD alone or fusion, and Gal4p AD alone or fusion were spotted on SD-Trp-Leu and SD-Trp-Leu-His (in the absence or presence of aminotriazole, AT) plates and incubated until visible colonies were formed.

Mentions: In a systematic analysis of protein complexes in yeast, Rpm2p was found in the same complex with Dcp2p by affinity capture using Dcp2p as bait in two independent attempts (30,31). Dcp2p is a subunit of the decapping enzyme and can be found in cytoplasmic processing bodies (32,31). To determine whether Rpm2p and Dcp2p interact in vivo, we performed the yeast two-hybrid analysis using Rpm2p as bait. We employed a reporter gene, HIS3, which is under control of the yeast GAL DNA-binding domain. To make the bait, we fused the RPM2 coding region lacking the first 41 amino acids downstream of the GAL DNA-binding domain (BD-Rpm2p). We introduced this plasmid into a reporter strain together with another plasmid expressing either Dcp2p fused to the GAL DNA-activation domain (AD-Dcp2p) or GAL DNA-activation domain alone (AD). Transformants were spotted on selective plates and scored for the ability to grow on medium lacking histidine and containing 3-aminotriazole (AT), a competitive inhibitor of the HIS3 gene product. Figure 2 shows that cells expressing both the AD-Dcp2p and BD can grow on medium lacking histidine, but growth ceases in the presence of 1 mM AT. This background growth is likely attributed to the leaky expression of the HIS3 gene. Cells expressing BD-Rpm2p and AD could grow in the presence of 1mM AT, however their growth was diminished in the presence of 10 mM AT and completely abolished at 15 mM AT. This is because Rpm2p itself has a transactivation domain containing two putative leucine zippers, and shows robust reporter activity in the absence of the carboxy-terminal domain, which is not required for growth by fermentation (8). However, in the presence of an intact carboxy-terminus in the construct used here, Rpm2p transactivation is moderate and can be abolished at lower concentrations of AT than used in a previous study (8). In contrast, cells expressing both, BD-Rpm2p and AD-Dcp2p show increased fitness compared to cells expressing BD-Rpm2p and AD, at all concentrations of AT tested. This result indicates that Rpm2p and Dcp2p can interact in vivo and substantiates a model that assigned Rpm2p as the attachment protein together with Xrn1p (a 5′ to 3′ exonuclease) and a protein of unknown function, Ybr094p, to a module (Edc3p-Dcp1p-Dcp2p) known as the mRNA-decapping complex (Gavin et al. (36)).Figure 2.


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 interacts with Dcp2p in the yeast two-hybrid assay. Yeast CG1945 strains expressing the combination Gal4p BD alone or fusion, and Gal4p AD alone or fusion were spotted on SD-Trp-Leu and SD-Trp-Leu-His (in the absence or presence of aminotriazole, AT) plates and incubated until visible colonies were formed.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 2: Rpm2p interacts with Dcp2p in the yeast two-hybrid assay. Yeast CG1945 strains expressing the combination Gal4p BD alone or fusion, and Gal4p AD alone or fusion were spotted on SD-Trp-Leu and SD-Trp-Leu-His (in the absence or presence of aminotriazole, AT) plates and incubated until visible colonies were formed.
Mentions: In a systematic analysis of protein complexes in yeast, Rpm2p was found in the same complex with Dcp2p by affinity capture using Dcp2p as bait in two independent attempts (30,31). Dcp2p is a subunit of the decapping enzyme and can be found in cytoplasmic processing bodies (32,31). To determine whether Rpm2p and Dcp2p interact in vivo, we performed the yeast two-hybrid analysis using Rpm2p as bait. We employed a reporter gene, HIS3, which is under control of the yeast GAL DNA-binding domain. To make the bait, we fused the RPM2 coding region lacking the first 41 amino acids downstream of the GAL DNA-binding domain (BD-Rpm2p). We introduced this plasmid into a reporter strain together with another plasmid expressing either Dcp2p fused to the GAL DNA-activation domain (AD-Dcp2p) or GAL DNA-activation domain alone (AD). Transformants were spotted on selective plates and scored for the ability to grow on medium lacking histidine and containing 3-aminotriazole (AT), a competitive inhibitor of the HIS3 gene product. Figure 2 shows that cells expressing both the AD-Dcp2p and BD can grow on medium lacking histidine, but growth ceases in the presence of 1 mM AT. This background growth is likely attributed to the leaky expression of the HIS3 gene. Cells expressing BD-Rpm2p and AD could grow in the presence of 1mM AT, however their growth was diminished in the presence of 10 mM AT and completely abolished at 15 mM AT. This is because Rpm2p itself has a transactivation domain containing two putative leucine zippers, and shows robust reporter activity in the absence of the carboxy-terminal domain, which is not required for growth by fermentation (8). However, in the presence of an intact carboxy-terminus in the construct used here, Rpm2p transactivation is moderate and can be abolished at lower concentrations of AT than used in a previous study (8). In contrast, cells expressing both, BD-Rpm2p and AD-Dcp2p show increased fitness compared to cells expressing BD-Rpm2p and AD, at all concentrations of AT tested. This result indicates that Rpm2p and Dcp2p can interact in vivo and substantiates a model that assigned Rpm2p as the attachment protein together with Xrn1p (a 5′ to 3′ exonuclease) and a protein of unknown function, Ybr094p, to a module (Edc3p-Dcp1p-Dcp2p) known as the mRNA-decapping complex (Gavin et al. (36)).Figure 2.

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