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. 2009 Nov 24;106(47):19848-53.
doi: 10.1073/pnas.0910754106. Epub 2009 Nov 10.

Localization of mRNAs coding for peroxisomal proteins in the yeast, Saccharomyces cerevisiae

Gadi Zipor  1 , Liora Haim-Vilmovsky, Rita Gelin-Licht, Noga Gadir, Cecile Brocard, Jeffrey E Gerst
Affiliations

Localization of mRNAs coding for peroxisomal proteins in the yeast, Saccharomyces cerevisiae

Gadi Zipor et al. Proc Natl Acad Sci U S A. .

Abstract

Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all ( approximately 50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11-15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%-80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%-42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed <<30% colocalization. mPP association with the peroxisome or ER was verified using cell fractionation and RT-PCR analysis. A model mPP, PEX14 mRNA, was found to be in close association with peroxisomes throughout the cell cycle, with its localization depending in part on the 3'-UTR, initiation of translation, and the Puf5 RBP. The different patterns of mPP localization observed suggest that multiple mechanisms involved in mRNA localization and translation may play roles in the importation of protein into peroxisomes.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Localization of endogenous mRNAs encoding peroxins. (A) Representative fluorescence microscopy images of cells bearing the MS2L sequence integrated into different genes (as indicated; see the ORFINTstrains in Table S2) and transformed with plasmids expressing MS2-CP fused with 3 GFP molecules [MS2-CP-GFP(x3)] and RFP-PTS1, as a marker for the peroxisomes, are shown. Cells were grown overnight on medium containing oleate and induced with the same medium lacking methionine for 1 h before visualization. The percentage of GFP-labeled RNA granules that colocalize with peroxisomes is shown. mRNA indicates labeled RNA granules; RFP-PTS1 indicates peroxisomes. (Scale bar: 2 μm). (B) Integration of the MS2 loops does not alter protein function. MS2L-integrated yeast strains (as indicated) were grown to log phase on glucose-containing medium, normalized for cell number, diluted serially, and plated by drops onto solid medium containing oleate.
Fig. 2.
Fig. 2.
Localization of endogenous mRNAs encoding matrix proteins. (A) Representative fluorescence microscopy images of cells bearing the MS2L sequence integrated into different genes (as indicated; see the ORFINTstrains in Table S2) and transformed with plasmids expressing MS2-CP-GFP(x3) and RFP-PTS1. Cells were grown on medium containing oleate and induced with the same medium lacking methionine for 1 h before visualization. The percentage of GFP-labeled RNA granules that colocalize with peroxisomes is shown. mRNA indicates labeled RNA granules; RFP-PTS1 indicates peroxisomes. (Scale bar: 2 μm). (B) PEX3 mRNA localizes to the ER. Representative fluorescence microscopy images of MS2L-tagged PEX3 cells transformed with plasmids expressing MS2-CP-GFP(x3) and Sec63-RFP (an ER marker). mRNA indicates labeled RNA granules; protein indicates ER labeled with Sec63-RFP. The percentage of GFP-labeled granules that colocalize with ER is shown. (C) Subcellular fractionation. Yeast expressing Sec63-GFP was fractionated by density gradient centrifugation into ER and cytoplasmic fractions (same fractions as shown in fig. 9b in ref. 7). RT-PCR was performed on DNase-treated RNA derived from these fractions using PEX3-specific primers. PCR samples were then electrophoresed and visualized on a 1% agarose gel. (See fig. 9b in ref. for other RNAs detected using the same conditions.)
Fig. 3.
Fig. 3.
Examination of mPP localization using cell fractionation and RT-PCR. (A) Western blot analysis of peroxisome purification. Peroxisomes were purified using density gradient centrifugation followed by affinity purification. Samples (40 μg) from the 3 fractions (PNS, postnuclear supernatant; 10%/35%, mitochondria-enriched membrane fraction; Perox, purified peroxisomes) were electrophoresed on SDS/PAGE gels and analyzed by immunoblotting. Antibodies (1:5,000) against Pot1 (peroxisome), Sed5 (Golgi), Nyv1 (Vacuole), and Sso1 (PM) were used for the chemiluminescent detection of proteins. (B) RT-PCR analysis of purified peroxisomes. Yeast cells were fractionated, and samples from the PNS, 10%/35%, and peroxisomal fractions were collected, from which RNA was purified. After DNase treatment and reverse transcription (RT; +), PCR was performed using gene-specific primers (as indicated). Samples without RT (-) were used as controls for DNA contamination. After PCR, samples were electrophoresed on agarose gels and documented. PEX1 and SEC61 mRNAs were detected in a parallel experiment.
Fig. 4.
Fig. 4.
Localization of PEX14 mRNA. (A) Illustration of the different MS2L-tagged PEX14 mRNAs. Three different MS2L-tagged PEX14 constructs— native, no 3′-UTR, and mutated ATG—all expressed under the MET25-inducible promoter, were used. (B) PEX14 mRNA associates with peroxisomes throughout the cell cycle. Representative time-lapse microscopy images of cells expressing tagged native PEX14 mRNA, as well as MS2-CP fused with 1 molecule of GFP (MS2-CP-GFP) and RFP-PTS1, are shown. Cells were grown on glucose-containing medium, and images were obtained with a DeltaVision imaging system at different times (minutes). For a series of deconvoluted images, see Movie S1. (C) Removal of the 3′-UTR or ATG diminishes PEX14 mRNA localization to peroxisomes. Representative images of cells expressing the tagged PEX14 mRNAs (i.e., native, no 3′-UTR, and mutated ATG) cotransformed with plasmids expressing MS2-CP-GFP(x3) and RFP-PTS1 are shown. Cells were grown overnight on medium containing oleate and shifted to the same medium lacking methionine for 1.5 h. The percentage of cells showing mRNA colocalization with peroxisomes is indicated. (D) Loss of PUF5 expression diminishes PEX14 mRNA localization to peroxisomes. Representative images show cells expressing tagged native PEX14 mRNA, MS2-CP-GFP(x3), and RFP-PTS1 in GAL1-PUF5 cells grown overnight on medium containing oleate and shifted to the same medium lacking methionine for 1.5 h. mRNA indicates GFP-labeled RNA granules; RFP-PTS1 indicates peroxisomes.
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