Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Podsumowanie

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Streszczenie

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes’ association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Wprowadzenie

Eukaryotic cells are organized in distinct compartments having specific functions. To accomplish its function, each compartment contains a unique set of proteins that are essential for its activity. A possible mechanism through which these proteins approach their compartment is by localized translation^1,2. In this process, the protein is synthesized at its destination by ribosomes and mRNAs that are located there. Among the probable advantages of localized translation are increased efficiency of protein targeting, decreased need for protein chaperones, and enabling site-specific regulation mechanisms. Also, localized mRNAs and ribosomes can be a secluded reservoir of translation machinery in cases of cellular stress, when general translation is inhibited.

Mitochondria became in recent years a central model to study localized translation.  Most mitochondria proteins are encoded in the nucleus, translated in the cytosol, and imported into the organelle. Various lines of evidence indicate that many of these proteins are produced through a local translation process. Initially, electron microscopy and biochemical fractionation studies detected ribosomes associated with mitochondria^3-5. These studies where then corroborated in vivo by work on specific mRNAs, that were found to be imported only in a cotranslational manner^6,7. Genome-wide studies of mRNAs association with mitochondria revealed that a significant fraction of mRNAs are localized to the mitochondria vicinity^8-10. Some of these mRNAs were further characterized by in vivo fluorescence methods, such as FISH or mTAG^9,11.  A straight-forward interpretation of this association is that these mRNAs serve as templates for localized translation.

The mechanisms by which these mRNAs approach the mitochondria are unknown. Noncoding domains (most significantly 3’ UTRs) were shown to be involved in mRNA association to the mitochondria¹². These domains are likely to serve as a binding site to RNA-binding proteins which mediate their transport. Studies in yeast revealed that a member of the PUM family of proteins (Puf3) supports mRNA association with mitochondria^8,13. A plausible role for Puf3, which is based on functions of other family members, is to inhibit translation while the mRNA is en route¹⁴. Thus, mRNAs may be transported in a nontranslated status, by RNA binding proteins that interact with noncoding regions. Alternatively, a large body of work suggests that transport occurs while the protein is being synthesized. In particular, translation inhibitors were shown to affect mRNAs association^8,13. Furthermore, translated features such as the AUG, mitochondrial targeting sequence (MTS) or ORF regions were shown to assist in localization^8,11,15. Hsp70-family protein chaperone and protein receptor on the mitochondria outer membrane were also shown to support mRNA association, further implying that encoded-protein features are important for mRNA localization¹⁶. This is consistent with a model in which the ribosome-emerging protein serves as recognition element for targeting mRNA-ribosome-protein complex to the mitochondria¹⁷.

Localized translation near the mitochondria was studied by various methods, including electron microscopy (to visualize ribosomes)³,  FISH⁹,  green RNA (to detect specific mRNAs)¹¹, and biochemical fractionation (to detect both RNA and ribosomes)^10,18. While the former methods detect localization in vivo and may allow visualization of transport dynamics, the latter allows detection of many different mRNAs in a single experiment. Furthermore, for biochemical fractionation coding or noncoding domains do not need to be altered, therefore their specific roles can be evaluated. Biochemical fractionation has been successfully used for many years, for isolation of many different cellular compartments. Its principals and limitations are well established, and one can easily modify existing protocols for different purpose. The necessary instrumentation is standard in many labs, therefore it is usually the first method of choice for studying intracellular localization. We describe a protocol that was optimized for isolation of mRNAs while ribosomes are associated with mitochondria. This protocol is therefore optimal for studying factors involved in localized translation near mitochondria.

Protokół

1. Mitochondria Purification

Weigh the pellet of cells. Roughly 0.6 g are obtained from 100 ml cells.

1. Grow 100-150 ml of yeast cells to OD₆₀₀ = 1-1.5 at 30 °C on a nonfermentable growth medium, such as galactose-based growth medium, in order to enrich for mitochondria.
2. Centrifuge cells at 3,000 x g for 5 min at room temperature and discard the supernatant.
3. Wash the pellet with double distilled water and centrifuge again. Discard the supernatant.
4. Resuspend the pellet in 1 ml DTT Buffer per 0.5 g of cells. It is important to treat the cells with a reducing agent in order to break the disulfide bonds within the cell wall, thereby improving hydrolysis.
5. Incubate the cells for 10 min at 30 °C with gentle shaking. Meanwhile, weigh 6 mg zymolyase per 1 g of cells and suspend it in Zymolyase Buffer.
6. Centrifuge cells at 3,000 x g for 5 min at room temperature and discard the supernatant.
7. Resuspend the pellet in 1 ml Zymolyase Buffer per 0.15 g of cells. Do not vortex.
8. Measure OD₆₀₀ of 10 μl aliquot of the cells in 990 μl of water.
9. Add Zymolyase (step 1.5) to the cells to hydrolyze glucose polymers at the β-1,3-glucan linkages and generate spheroplasts. From this step, spheroplasts should be kept in an isotonic solution in order to avoid lysis.
10. Incubate cells for 15 min at 30 °C (for optimal activity of zymolyase) with gentle shaking. To verify hydrolysis of the cell wall and spheroplasts generation, mix 10 μl of the cells with 990 μl of water. Spheroplasts are expected to lyse due to osmotic difference, and the OD₆₀₀ should be at least 10-fold less than the value determined in step 1.9. If not, continue incubation with zymolyase for another 15 min.
11. Centrifuge cells at 3,000 x g for 5 min at room temperature. Carefully discard the supernatant, as the pellet might be unstable.
12. Wash spheroplasts with Zymolyase Buffer and discard the supernatant.
13. Resuspend spheroplasts with 100 ml Recovery medium. Transfer spheroplasts to an Erlenmeyer flask and incubate for 1 hr at 30 °C with shaking. This recovery step is necessary since during the Zymolyase treatment translation is arrested and mRNA localization is disrupted (Figure 1B).
14. Add 0.1 mg/ml CHX and transfer spheroplasts to a precooled 50 ml conical tube. CHX addition is important to freeze ribosomes’ association with mRNAs. Thus, ribosomes-dependent mRNA association with mitochondria is maintained.
15. Centrifuge spheroplasts at 3,000 x g for 5 min at 4 °C and discard the supernatant.
16. Wash twice with cold Mannitol Buffer.
17. Resuspend spheroplasts with 4 ml cold Mannitol Buffer and transfer them to a Dounce homogenizer of 15 ml capacity equipped with tight fitting pestle. Gently break spheroplasts with 15 strokes and transfer the lysate to a 13 ml tube.
18. Centrifuge the lysate at 1,500 x g for 6 min at 4 °C to pellet nuclei and unbroken cells.
19. Carefully transfer the supernatant to a new tube. Set aside 1 ml (25%) of the sample as an unfractionated sample (“Total” sample). Transfer 50 ml of this sample to a new tube and add 15 ml of 4X LSB for western blot analysis.  Precipitate RNA from the rest of the “Total” sample (See Protocol 3, RNA precipitation).
20. Centrifuge the supernatant at 10,000 x g for 10 min at 4 °C to pellet the mitochondria.
21. Transfer the supernatant (~3 ml) to a new tube and keep it on ice. This is the “Cytosolic” fraction. Transfer 50 ml of this sample to a new tube and add 15 ml of 4x LSB for western blot analysis. Precipitate RNA from the rest of the “Cytosolic” sample (See Protocol 3, RNA precipitation).
22. Wash the pellet with 3 ml Mannitol Buffer and centrifuge again at 10,000 x g for 10 min at 4 °C.
23. Resuspend the pellet with 3 ml Mannitol Buffer. This sample contains the mitochondria, and referred to as “Mitochondria” fraction. Transfer 50 ml of this sample to a new tube and add 15 ml of 4x LSB for western blot analysis.

2. RNA Extraction

1. Add to each sample one volume of 8 M guanidinium-HCl and two volumes of 100% ethanol. Vortex and incubate for at least 2 hr at -20 °C.
2. Centrifuge samples at 10,000 x g for 20 min at 4 °C. Discard supernatant. Be careful as the pellet might be unstable.
3. Wash the pellet with 70% EtOH.
4. Resuspend the pellet with 400 ml of RNase-free water and transfer the sample to a new Eppendorf tube.
5. Precipitate the RNA again by adding 0.1 volume of 3 M sodium acetate pH 5.2 and two volumes of 100% EtOH. Vortex and incubate for at least 2 hr at -20 °C.
6. Centrifuge samples at 20,000 x g for 20 min at 4 °C. Discard the supernatant and wash with 70% EtOH.
7. Air dry the pellet and resuspend with RNase-free water. Store RNA samples at -80 °C. Samples can be used for northern analysis¹⁹ or microarray analysis¹⁸ (See Protocol 3).

3. Preparing the RNA for Microarray Analysis

1. Resuspend the mRNA from step 2.2 with 650 ml of RNase-free water.
2. To remove any residual DNA or proteins, add equal volume (650 ml) of acidic phenol:chloroform (5:1, pH 4.7) and vortex vigorously. Centrifuge at maximum speed for 2 min.
3. Transfer 500 ml of the upper phase (the aqueous phase) to a new tube. Avoid taking the interphase, as it contains DNA. Also be careful of taking phenol, which can inhibit the reverse transcription reaction.
4. The RNA sample contains a significant amount of heparin, which is a potent inhibitor of reverse transcriptase. Thus, for RT-PCR or microarray labeling it needs to be removed. Remove heparin by LiCl precipitation: add LiCl to a final concentration of 2 M and incubate samples over night at -20 °C.
5. Thaw the samples at 4 °C and centrifuge at 20,000 x g for 20 min at 4 °C.
6. Carefully discard the supernatant and wash the pellet with 80% ethanol. Centrifuge again as described in step 3.5.
7. Discard the supernatant, air dry and resuspend the pellet in 150 ml of RNase-free water.
8. To remove any residual LiCl, precipitate again with 0.1 volume of 3 M sodium acetate pH 5.3 and 3 volumes of 100% ethanol. Incubate at -20 °C for at least 2 hr.
9. Centrifuge at top speed for 20 min at 4 °C. Wash carefully the transparent pellet with 80% ethanol and air dry.
10. Resuspend the pellet with 25 ml RNase-free water and keep the samples at -80 °C.
11. Follow the steps for RNA labeling and hybridization described in^18,20.

Wyniki

This protocol allows separation of a mitochondria-containing fraction from cytosolic components. The best way to test its success is to perform northern analysis and western analysis (Figure 1) to samples from the different isolation steps. Three parameters for the isolation quality are derived from these analyses. First, whether the RNA or proteins in the samples are intact – these will be detected as distinct bands in the analyses. Degradation events will induce the appearance of additional, shor...

Dyskusje

Technological advancements in imaging had yielded high resolution tools to study mRNA localization. Today, one can measure the movement of even a single mRNA molecule at the msec time scale^23-26. Yet, traditional biochemical approaches, as the one described above, are also informative and are preferable in some cases. Biochemical isolation allows purification of a large repertoire of mRNAs and proteins, and is therefore preferable for genome-wide studies. In a single isolation, one can obtain sufficient mRNA l...

Materiały

Name	Company	Catalog Number	Comments
Yeast Extract
BactoPeptone
Galactose			Do not autoclave Galactose
Growth medium			For mitochondrial enrichment, you should use any nonfermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose
0.1 M Tris-HCl, pH 9.4
30 mM Tris-HCl, pH 7.6
Dithiothreitol (DTT)
10 mM DTT Buffer			0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time
1.2 M Sorbitol
4 mM KH2PO4
16 mM K2HPO4
0.2 μm filter
Zymolyase Buffer			1.2 M Sorbitol, 4 mM KH2PO4 , 16 mM K2HPO4. Filter this buffer (0.2 μm) and keep at room temperature for future use
Zymolyase 20T			20,000 U/g
Recovery medium			Galactose-based growth medium supplemented with 1 M Sorbitol
0.1 mg/ml Cycloheximide (CHX)
0.6 M Mannitol
5 mM MgAc
100 mM KCl
0.5 mg/ml Heparin
1 mM Phenylmethanesulfonylfluoride (PMSF)
Mannitol Buffer			0.6 M Mannitol, 30 mM Tris-HCl pH7.6, 5 mM MgAc, 100 mM KCl. Add freshly: 0.5 mg/ml Heparin, 0.1 mg/ml CHX and 1mM (PMSF). Filter this buffer and use it ice-cold
8 M Guanidinium-HCl
100% and 70% Ethanol (EtOH)
3 M Sodium Acetate, pH 5.2
10 M LiCl stock solution
250 mM Tris HCl pH 6.8
SDS
Glycerol
β-Mercaptoethanol
Bromophenol blue
4x LSB			250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% β-mercaptoethanol, and 0.02% Bromophenol blue
Dounce homogenizer of 15 ml capacity equipped with tight fitting pestle