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

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.

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.

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.

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.

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.

Figure 5.

Inhibition of protein synthesis by cycloheximide. Equal numbers of mid-log phase cells, expressing either Dcp2p-RFP or both Dcp2p-RFP and GFP-Rpm2p, pretreated for 30 min with 100 μg/ml cycloheximide, were incubated with ³⁵S-labeled methionine for 30 min, without (open bars) or with (black bars) cycloheximide pretreatment for 30 min. TCA-insoluble material was measured by scintillation counting. Columns represent an average mean of the representative experiment performed in triplicate of three biological samples.

Figure 6.

P bodies are affected by proteasome mutants. The wild-type strain, a strain carrying a mutation in UMP1, and a strain lacking RPM2 but carrying a mutation in the PRE4 all expressing Dcp2p-RFP were grown in glucose synthetic medium and P bodies visualized before and after incubation with cycloheximide for 30 min.

Figure 7.

Depletion of ubiquitin in cells expressing GFP-Rpm2p. (A) Cells expressing either Dcp2p-RFP or Dcp2p-RFP and GFP-Rpm2p were grown in synthetic galactose medium in the exponential phase, and total protein extracts were analyzed by Western analysis with anti-ubiquitin antibodies after gel-separation and transfer to the membrane. Increasing amounts of protein extracts were used: 2 μg, lanes 1 and 2; 4 μg, lanes 3 and 4; 8 μg, lanes 5 and 6. *, unknown protein recognized by anti-ubiquitin antibodies serves as a loading control. A part of the membrane where free ubiquitin is detected was underexposed (bottom panel). (B) The same membrane was reprobed with anti-GFP antibodies.

Figure 8.

Kinetics of the induction and localization of GFP-Rpm2p fusion protein. (A) Western analysis of GFP-Rpm2p fusion protein before and after addition of galactose. The protein was detected using both anti-GFP and Rpm2p antibodies. Rpm2p shows the migration of endogenous Rpm2p. X, unknown protein recognized by anti-Rpm2p antibodies serving as a loading control. (B) Kinetics of GFP-Rpm2p colocalization with Dcp2p-RFP before and after addition of galactose.

Figure 9.

GFP-Rpm2p localization when expressed from its endogenous promoter. GFP-Rpm2p is observed in P bodies in the absence but not the presence of cycloheximide (CHX) (two upper panels). Localization of GFP-Rpm2p to P bodies does not require Lsm1p or Xrn1p, other components of P bodies (two bottom panels). The Dcp2p-RFP was used as a P body marker in colocalization studies.