Abstract
Targeted mRNA localization to distinct subcellular sites occurs throughout the eukaryotes and presumably allows for the localized translation of proteins near their site of function. Specific mRNAs have been localized in cells using a variety of reliable methods, such as fluorescence in situ hybridization with labeled RNA probes, mRNA tagging using RNA aptamers and fluorescent proteins that recognize these aptamers, and quenched fluorescent RNA probes that become activated upon binding to mRNAs. However, fluorescence-based RNA localization studies can be strengthened when coupled with cell fractionation and membrane isolation techniques in order to identify mRNAs associated with specific organelles or other subcellular structures. Here we describe a novel method to isolate mRNAs associated with peroxisomes in the yeast, Saccharomyces cerevisiae. This method employs a combination of density gradient centrifugation and affinity purification to yield a highly enriched peroxisome fraction suitable for RNA isolation and reverse transcription-polymerase chain reaction detection of mRNAs bound to peroxisome membranes. The method is presented for the analysis of peroxisome-associated mRNAs; however it is applicable to studies on other subcellular compartments.
Key words
1 Introduction
A number of studies over the past two decades have demonstrated that mRNAs can undergo targeting to specific subcellular sites in eukaryotic cells in order to regulate cell division, cell polarity and differentiation, and body plan development in metazoans (1–3). Thus, mRNA targeting is now considered to be a widespread phenomenon that occurs in unicellular organisms, animal and plant tissues, and in developing embryos from a variety of animal phyla (2–4). The transport and localization of mRNAs within the cell can be achieved by different mechanisms, such as local RNA synthesis, local protection from degradation, diffusion and anchoring, or active transport by molecular motors (5).
Because of the importance of mRNA localization in the correct placement of proteins within cells, there is a need for specialized tools to monitor mRNA trafficking and localization in individual cells using fluorescence microscopy. The most commonly used technique is fluorescence in situ hybridization (FISH); a method that uses short DNA or RNA probes, that either bear fluorophores or can be detected using fluorescent-labeled antibodies, to hybridize to denatured RNA within cells. The advantage of FISH is that it can detect the endogenously expressed mRNAs, however, this method necessitates the use of fixed material and does not allow for the monitoring mRNAs in vivo. A tool that is commonly used for the localization of mRNAs in vivo is the bacteriophage MS2 coat protein (MS2-CP) that can bind to a short RNA stem-loop sequence (the MS2 aptamer) inserted into an mRNA of interest. When fused with a fluorescent protein, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), the MS2-CP reporter can visualize mRNAs tagged with the MS2 loop sequences (6). A more recent application of this method is m-TAG (7, 8), a gene-tagging procedure which allows the sustained visualization of endogenously expressed mRNAs in living yeast. This procedure uses a PCR-based strategy for insertion of the MS2 loops into any gene of interest in the genome by homologous recombination. Upon co-expression of MS2-CP fused with GFP(3×), it is possible to examine the localization of endogenous mRNAs in vivo. This technique has been successfully used to localize specific mRNAs (many for the first time) to the endoplasmic reticulum (ER), mitochondria, peroxisomes, and to the bud tip (7). While the advantage of FISH and m-TAG is that both methods can efficiently localize endogenous mRNAs, their weakness is that by focusing on specific genes they are less applicable for rapid large-scale studies aimed at visualizing all mRNAs that localize to a given intracellular structure.
In contrast, a method that enables mRNA localization on a larger scale is subcellular fractionation, which yields purified organelles/membranes that can be assayed by reverse transcription-polymerase chain reaction (RT-PCR) with specific oligonucleotides or using DNA microarrays to detect mRNAs in the purified fractions. For example, microarray analyses on preparations derived from both yeast and mammalian cells revealed that thousands of mRNAs, including those coding for cytosolic proteins, are enriched in ER membranes (9, 10). Likewise, others have shown that mRNAs encoding mitochondrial proteins of prokaryotic origin preferentially localize to the vicinity of mitochondria (11) and that >500 mRNAs associate with mitochondria in yeast (12). In the procedure outlined below, we describe a method to obtain a highly enriched peroxisome fraction, using subcellular fractionation followed by affinity purification, and identify the associated mRNAs using RT-PCR. Application of this procedure has successfully demonstrated that mRNAs encoding certain peroxisomal proteins localize to peroxisome membranes (13).
Peroxisomes are single membrane-bound organelles containing diverse enzymes related to lipid metabolism and are found exclusively in eukaryotes (14–17). In yeast, peroxisome function is essential for the catabolism of fatty acids, which makes this organism useful for the study of peroxisome biogenesis (18). While many studies have explored the mechanism by which proteins are imported into the peroxisome matrix (19), less is known of how peroxisome membrane proteins and peroxins, the proteins involved in peroxisome biogenesis (20), are imported. The targeted transport of mRNA to the peroxisome membrane may confer localized synthesis near the import machinery and, thus, facilitate protein translocation into the organelle.
1.1 Overview of the mRNA Isolation Method Using Cell Fractionation and Affinity Purification
The principles of the peroxisome purification method are illustrated in Fig. 1. The basic procedure includes the separation of organelles using isopycnic centrifugation on a Nycodenz gradient, followed by affinity purification. Peroxisomes are specifically purified from the gradient fraction using an epitope-tagged anchor protein. Briefly, an HA epitope-encoding sequence was appended at the 3′end of the PEX30 locus in wild type yeast cells to allow for endogenous expression of the HA-tagged Pex30 peroxisomal membrane protein. Peroxisomes were then purified from the enriched gradient fraction using agarose beads conjugated with anti-HA antibodies. After purification, RNAs were isolated from the purified peroxisomes and assayed by RT-PCR using oligonucleotide pairs to specific genes.
In yeast, peroxisomes are the only site for the β-oxidation of fatty acids. Therefore peroxisome function is required for cells to grow on medium containing fatty acids as the sole carbon source. First (see Subheading 3, step 1), yeast cells expressing HA-Pex30 are pre-grown to log phase on glucose-containing medium, followed by overnight culture in oleate-containing medium to induce peroxisome proliferation. Second (step 2), cells are harvested, washed, and treated with Zymolase (yeast lytic enzyme) to remove the cell wall and create spheroplasts. Next (step 3), the spheroplasts are lysed and homogenized using a Dounce homogenizer. The lysed spheroplasts are then centrifuged and the post-nuclear supernatant (PNS) obtained is loaded on top of a Nycodenz step density gradient (step 4) and further centrifuged to yield an enriched peroxisome fraction that is separated from the cytosol and other membrane-containing fractions, such as the mitochondria. Next (step 5), the peroxisome fraction is further purified using agarose beads conjugated to anti-HA antibodies. Both total RNA and protein are then extracted (step 6) from a sample of the PNS, the membrane/mitochondrial fraction, and the purified peroxisome fraction. The purity of the peroxisome fraction is verified by western analysis using specific antibodies against ER, Golgi, plasma membrane, cytosolic, and peroxisomal proteins, while mRNAs present in the peroxisome fraction are examined using either semi-quantitative or real-time PCR. See ref. 13 for examples of western and PCR analysis.
2 Materials
2.1 Cell Culture and Harvesting
-
1.
SC medium: 1 l liquid medium is composed of 7 g of Synthetic Dry Mix [mix composed of: 294 g ammonium sulfate; 30 g dibasic potassium phosphate; 0.3 g each of arginine, cysteine, and proline; 0.45 g each of isoleucine, lysine, and tyrosine; 0.75 g each of glutamic acid, phenylalanine, and serine; 1.0 g each of aspartate, threonine, and valine; and 100 g Yeast nitrogen base lacking ammonium sulfate and amino acids (Becton, Dickinson and Co., cat. no. 233520)], 10 ml amino acid stock mix [200 ml of 100× synthetic complete medium composed of: 0.4 g each of adenine, uracil, tryptophan, histidine, methionine, and leucine in 150 ml DDW; acidify with 3 ml concentrated HCl; fill to 200 ml with DDW], 5 g or 20 g glucose (as required), 0.35 ml 10 N NaOH; and DDW to 1 l (without diethylpyrocarbonate; DEPC); final pH = 6–7. For further details on preparation, see recipe in Haim-Vilmovsky and Gerst (8).
-
2.
Oleate mix: 10% (vol/vol) oleic acid (Merck, cat. no. k3711571), 1% (vol/vol) Tween 80 (Sigma, cat. no. p8074), 17.7% (vol/vol) 1 N NaOH. Boil for 2 h in a water bath and again for 1 h before each use (to emulsify and sterilize).
-
3.
Oleate induction medium: 0.3% (wt/vol) Yeast extract (Becton, Dickinson and Co., cat. no. 212750), 0.5% (wt/vol) Peptone (Becton, Dickinson and Co., cat. no. 211677), 0.5% (wt/vol) KH2PO4, and adjust solution to pH 6 with 1 M K2HPO4; autoclave for 30 min and add 2% oleate mix after cooling.
-
4.
Ultra-pure water or DEPC-treated DDW.
-
5.
Tris–sulfate 1 M, pH 9.4.
-
6.
Sorbitol/Potassium phosphate buffer: 1.2 M Sorbitol and 20 mM Potassium phosphate buffer at pH 6.
-
7.
Dithiothreitol (DTT).
2.2 Spheroplasting
-
1.
Yeast lytic enzyme (Zymolase); 77,200 U/g (ICN Biomedicals, cat. no.152270).
-
2.
2% (wt/vol) Sodium dodecyl sulfate (SDS) in DDW.
2.3 Homogenization
-
1.
PEG/Sucrose Buffer (PSB): 5 mM 2-(N-morpholino)ethanesulfonic acid (Mes), 1 mM KCl, 0.5 mM EDTA (adjusted to pH 6.0 with KOH), 12% (wt/vol) PEG 1500, and 160 mM Sucrose. Add freshly made protease inhibitors and RNAsin [Final concentrations: 2 μg/ml aprotinin, 1 μg/ml pepstatin, 0.5 μg/ml leupeptin, 1 mM phenylmethanesulphonylfluoride (PMSF), and 40 U/ml RNAsin (Promega, cat. no. W251B)].
-
2.
Teflon dounce-type homogenizer (7 ml volume).
2.4 Nycodenz Layering and Density Gradient Centrifugation
-
1.
Nycodenz gradient: 35%, and 50% (wt/wt) Nycodenz (AG Axis shield PoC, cat. no. 1002424), refraction index: 1.4070, and 1.4280, respectively) in PSB. Before use add freshly made protease inhibitors + RNAsin (see Subheading 2.3, item 1) to the Nycodenz solutions.
-
2.
Ultracentrifuge, SW41-type rotor.
2.5 Affinity Purification
-
1.
Pull-Down Buffer (PDB): 50 mM Tris/HCl at pH 7.5, 1 mM DTT, 12% (wt/vol) PEG 1500, 160 mM sucrose, and freshly prepared protease inhibitors and RNAsin (see Subheading 2.3, item 1).
-
2.
Ultracentrifuge, SW60-type rotor.
-
3.
Monoclonal Anti-HA Agarose beads (Sigma, cat. no. A2095).
2.6 RNA Purification and RT-PCR
-
1.
MasterPure™ yeast RNA purification kit (Epicentre Biotechnologies, cat. no. MPY03100).
-
2.
Molony murine leukemia virus reverse transcriptase and RNase H minus (M-MLV H(−)RT; Promega, cat. no. M368B).
2.7 Western Blotting
-
1.
8% SDS–PAGE gel (mini).
-
2.
Protein sample buffer (1×): 80 mM Tris–HCl at pH 6.8, 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.25% (wt/vol) bromophenol blue.
-
3.
1% SDS.
3 Methods
3.1 Cell Culture and Harvesting
-
1.
Inoculate an overnight culture of yeast in 10 ml SC medium containing 2% (wt/vol) glucose.
-
2.
Next day, dilute cells to OD = 0.2 at A 600 in 1 l SC medium containing 0.5% (wt/vol) glucose and grow cells to OD = ∼2 at A 600.
-
3.
Spin down cells and inoculate to OD = 1 at A 600 in 1 l Oleate induction medium (harvest 500 ml of the SC medium OD = ∼2 at A 600, and resuspend with 1 l oleate medium).
-
4.
Incubate cells overnight at 30°C with shaking at ∼200 rpm.
-
5.
Harvest cells by centrifugation at 3,400 × g for 5 min using a Sorvall-type centrifuge with a rotor for use with large volumes (see Note 1).
-
6.
Wash 2× with DDW.
-
7.
Resuspend cell pellet with 0.1 M Tris/Sulfate at pH 9.4 and 10 mM DTT; Use 1 ml buffer for each 0.5 g of cells (i.e. 6 ml buffer for 3 g of cells).
-
8.
Incubate for 15 min at 30°C with gentle shaking.
-
9.
Spin down for 3 min at 1,400 × g at room temperature.
-
10.
Wash in SB; use 1 ml SB per gram of cells.
3.2 Spheroplasting
-
1.
Resuspend cell pellet at 0.15 g cells/ml SB containing Yeast lytic enzyme (10 mg/g cells).
-
2.
Incubate with gentle shaking at 30°C for 15–30 min.
-
3.
Microscopy: Verify spheroplast formation using 2% (wt/vol) SDS buffer (mix 2 μl of cells with 2 μl of 2% SDS buffer on a microscope slide); spheroplasts lyse upon contact with SDS-containing buffer and appear as dark spots under phase contrast, as opposed to intact cells which still bear their cell wall.
-
4.
Harvest spheroplasts using a Sorvall centrifuge with an SS34-type rotor at 1,500 × g for 5 min at 4°C (see Note 2).
-
5.
Wash 2× in SB using gentle resuspension.
3.3 Homogenization
-
1.
Gently resuspend spheroplasts in 1.5 ml/g cells with PEG/Sucrose Buffer (PSB) containing protease inhibitors + RNAsin. For example, for 3 g cells, add 4.5 ml PSB buffer (see Note 3).
-
2.
Homogenize using a motor-driven Potter-Elvehjem small clearance teflon pestle in a glass tube homogenizer at 1,500 rpm (use 20 slow strokes) on ice (see Note 4).
-
3.
Harvest nuclei/unbroken cells and cell debris using a Sorvall centrifuge with an SS34-type rotor for 5 min at 4°C at 1,400 × g (see Note 5). Remove soluble fraction (i.e. post-nuclear supernatant/PNS) to ice; save ∼30 μl for western analysis (see Note 6).
-
4.
Carefully add 50% Nycodenz (wt/wt in PSB; refraction index = 1.4280) to PNS to reach a final concentration of 10% (vol/vol) Nycodenz (see Note 7).
-
5.
Load the PNS (containing 10% Nycodenz) on top of a Nycodenz step gradient (see below) (see Note 8).
3.4 Nycodenz Layering and Density Gradient Centrifugation
-
1.
Layer the bottom of a 13-ml Beckman centrifuge tube (Polyallomer; cat. no. 331372 for use with a SW41-type rotor) with 2.5 ml of 50% (wt/wt) Nycodenz in PSB.
-
2.
Next gently layer 6.5 ml of 35% (wt/wt) Nycodenz in PSB on top of the 50% Nycodenz bottom layer. Use care not to disturb the layer.
-
3.
Finally, add 2.5 ml of the PNS in 10% (wt/wt) Nycodenz. Fill up the tubes with PNS containing 10% Nycodenz, if necessary.
-
4.
Centrifuge in an ultracentrifuge using an SW41-type rotor at ∼80,000 × g for 1 h at 4°C.
-
5.
Collect the visible cloudy peroxisome and membrane/mitochondria fractions (located just above the 50%:35% and 35%:10% interfaces, respectively) using a 2–5 ml syringe (needle gauge = 21 G, length = 0.8 × 40 mm). Usually ∼1–2 ml should be collected per fraction (see Note 9).
3.5 Affinity Purification
-
1.
Carefully dilute the peroxisome fraction with three volumes of PDB (containing proteases and RNasin). Add to a 3-ml Beckmann centrifuge tube (Polyallomer; cat. no. 355636).
-
2.
Pellet the peroxisomes using an ultracentrifuge with an SW60-type rotor. Spin at ∼80,000 × g for 20 min at 4°C.
-
3.
Decant supernatant and gently resuspend peroxisomes in 400 μl of PDB (containing proteases and RNasin).
-
4.
Divide the peroxisome fraction into two equal aliquots (∼200 μl each) and incubate each suspension with 25 μl anti-HA antibody cross-linked beads, pre-washed 3× in PDB as follows: add 50 μl aliquots of the 50% bead slurry to separate tubes before washing each bed with PDB; and add the peroxisome suspension directly to the washed beads.
-
5.
Incubate overnight at 4°C with rotation.
-
6.
Wash 3× for 30 min each with 200 μl PDB.
-
7.
Decant supernatant without removing beads.
3.6 RT-PCR and Western Analysis
To one aliquot perform RNA purification, reverse transcription, and PCR (steps 1–3). To the other aliquot perform western analysis (step 4).
-
1.
RNA purification:
We employ the MasterPure™ yeast RNA purification kit:
Add 300 μl of extraction reagent (containing Proteinase K) to the beads and boil for 15 min. Follow mRNA extraction instructions according to the protocol of the manufacturer (including the optional DNase treatment step).
Typically 500–1,000 ng of total RNA is obtained from the peroxisome fraction originating from 3 g of cells.
In parallel, use 200 μl of the PNS and 10%/35% fractions for mRNA purification: dilute the fractions 1:1 with DDW, add 300 μl extraction reagent (containing Proteinase K), and follow the mRNA extraction instructions.
-
2.
Reverse transcription (M-MLV H(−)RT):
Use 250–500 ng RNA from each fraction for each RT reaction. Use random hexamers (Fermentas, cat. no. S0142) and follow the manufacturer instructions, e.g. we use 10 min RT and 42°C for 50 min.
As a control, perform the same procedure without adding reverse transcriptase.
-
3.
PCR:
Semiquantitative PCR (between 18 and 24 cycles for detection) should be performed with gene-specific primers (∼20 bp; 10 pmol each per reaction) complimentary to a region of 300–500 nucleotides of the ORF. The PCR reaction is performed in a 20 μl reaction volume under standard conditions recommended by the manufacturer (i.e. we use 2× Taq Master mix purple; Lambda Biotech, cat. no. D123P; final (1×) composition: 1.5 mM MgCl2, 200 μM dNTPs, and 2.5 U/25 μl of taq polymerase). Use 1 μl of the reverse transcriptase reaction (e.g. cDNA) per PCR reaction.
PCR should be performed using the following conditions:
Melting temp – 95°C
Annealing temp – 55°C
Elongation temp – 72°C
Number of cycles typically employed for detection: 18, 21, and 24 cycles.
Load 20 μl of the PCR reactions onto a 1% (wt/vol) agarose gel in TAE buffer (1×); electrophorese at 120 V for ∼20 min. For an example of the expected results, see Fig. 3 in Zipor et al. (13) (see Note 10).
-
4.
Western analysis:
To the other aliquot of beads add 90 μl elution buffer (consisting of: 1× sample buffer containing 2% SDS and freshly added 10 μl of 1 M DTT), boil for 10 min and collect the soup; add and load aliquots of 25–30 μl onto a 8% SDS–PAGE mini-gel. Load PNS and 10%/35% fractions samples (e.g. Load 2–3 μl and ∼6 μl of the PNS and 10%/35% fractions, respectively). Assess the relative level of purity of the peroxisome fraction (relative to samples of the PNS and 10%/35% fractions) using specific antibodies against proteins of the peroxisome, Golgi, mitochondria, plasma membrane, cytoplasm, etc.
4 Notes
-
1.
Oleic acid-containing medium (oleate induction medium) is somewhat viscous and can make it more difficult to pellet the yeast cells, thus, the RPM used to pellet (Subheading 3.1, step 5) is higher than that typically used. Make sure that the size of the yeast pellet (Subheading 3.1, step 5) is sufficient for the procedure (obtain at least 3 ml of cells – i.e. ∼3 g of cells). If not, elevate the speed and time of centrifugation or increase the culture volume.
-
2.
Following zymolase treatment (Subheading 3.2), it is essential that all steps are performed on ice.
-
3.
It is very important to use RNase-free instruments and solutions. The process contains many steps and the amount mRNA obtained is small, so one should be careful with the handling of the material.
-
4.
Make sure that most of the cells are broken during the homogenization step (Subheading 3.3, step 2). If not, add several additional strokes and verify that the teflon pestle tightly fits the glass homogenizer.
-
5.
It is very important to properly remove the nuclei and cell debris after homogenization in order to avoid contamination of the PNS. If required, the PNS can be centrifuged in a new clean tube for another 15 min at 4°C using an SS34-type rotor at 1,400 × g.
-
6.
To monitor the degree of purification, we recommend removing aliquots at each step of purification; determine protein concentration and immediately add SDS-containing sample buffer and freeze until needed for analysis by western blotting.
-
7.
By no means should the PNS be centrifuged at high speed and separated into supernatant and pellets before the dilution with Nycodenz (and loading onto the gradient). High-speed centrifugation and pelleting in the absence of dilution can lead to contacts between organelles that remain following ultracentrifugation and, thus, lead to contamination of the peroxisome fraction.
-
8.
The objective of isopycnic centrifugation is to obtain an enriched peroxisome fraction that is separate from other organelles (i.e. mitochondria) and membranes, and has been established here for the use with Nycodenz only.
-
9.
The enriched peroxisome fraction collected after gradient centrifugation can be kept at 4°C for up to 24 h without noticeable protein degradation.
-
10.
The PCR reactions (Subheading 3.6, step 3) should show differences in the mRNA content between the various fractions. If no products are observed, one can increase the number cycles. Always include positive and negative controls for the PCR reactions. One can employ yeast genomic DNA as a template for the positive controls.
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Acknowledgements
This work was supported by grants to J.E.G. from the Minerva Foundation, Germany and Josef Cohn-Minerva Center for Biomembrane Research at the Weizmann Institute of Science, Israel. J.E.G. holds the Besen-Bender Chair in Microbiology and Parasitology. C.B. is supported by the Elise-Richter Program of the Austrian Science Fund (FWF).
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Zipor, G., Brocard, C., Gerst, J.E. (2011). Isolation of mRNAs Encoding Peroxisomal Proteins from Yeast Using a Combined Cell Fractionation and Affinity Purification Procedure. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_20
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DOI: https://doi.org/10.1007/978-1-61779-005-8_20
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