RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods
© de Heredia and Jansen; licensee BioMed Central Ltd. 2004
Received: 22 June 2004
Accepted: 01 October 2004
Published: 01 October 2004
The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.
We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins.
We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.
Genome analyses of a range of organisms have lead to the identification of an increasing number of putative RNA-binding proteins (RBPs) whose function is still unknown. RBPs have been found to act as integral part of ribonucleoparticles (RNPs) controlling gene expression at different levels . RNPs are involved in controlling RNA export, RNA stability, RNA subcellular localization and mRNA translation . It has been proposed that in this context RBPs could act as central coordinators in regulating the expression and fate of specific subsets of RNAs. This model is reminiscent of bacterial operons, where the expression of genes that act in the same pathway is regulated as one unit .
Recently, research has mainly been focused on identifying protein-protein interactions using two-hybrid interactions [4, 5], immunopurification  or affinity purification . So far, only few examples have been reported that were aimed at the identification of mRNA-protein interactions. In yeast, immunopurification has, for example, been used to enrich RNP complexes leading to the identification of 22 mRNAs localized to the bud tip , to the identification of Lhp1p associated mRNAs  and to the identification of mRNA export factor associated transcripts . There are many examples for affinity purification methods in yeast, but perhaps the one that has been used most extensively is the Tandem Affinity Purification (TAP). TAP consists of two serial affinity purification steps of a protein tagged with a double epitope tag, without affecting the expression level of the protein. It was first described for identifying new protein components of the yeast U1 snRNP  and later used to identify protein-protein interactions in yeast , bacteria , Trypanosoma brucei , Drosophila  and mammals . It has also been used to describe the set of mRNAs associated with the Puf family of RNA-binding proteins in yeast .
Besides the purification method, the way to lyse cells is also crucial. In yeast, different lysis methods are in use. Glass bead milling has been applied to identify RNAs from immunoprecipitated RNPs . Both French Press and glass bead milling have been successfully used to characterize protein-protein [6, 11] and protein-RNAs interactions . However, the integrity of the mRNA has not been determined under the conditions used. Here, we show that existing lysis methods lead to extensive mRNA degradation even in the presence of RNase inhibitors. We also present evidence that a third method, grinding deep-frozen cells at ultra-low temperature, can be used to obtain intact mRNAs.
Glass bead mill lysis leads to degraded RNAs
Breaking yeast with a glass bead mill is a common method to produce cell lysates. The principle is based on the physical rupture of the yeast's cell wall and cell due to the friction produced by glass beads rapidly moving through the cell suspension. One of the advantages of this method is the high lysis efficiency.
The chosen purification method involves several steps: lysis, extract preparation and IgG immunopurification. To rule out in which step RNA degradation takes place, we analysed mRNA integrity in samples taken at different purification steps (Figure 1C and data not shown). Therefore, we used a strain where Pbp2p, a putative RNA-binding protein that contains two KH-type 1 domains (hnRNP K homology domain) , has been tagged and a wild type strain. We found that extensive degradation already occurs during cell lysis (Figure 1C) as shown by Northern blot against a specific abundant mRNA (PDA1) or by direct staining of ribosomal RNAs after blotting with methylene blue (unbalanced ratio of 25S and 18S ribosomal RNAs in Figure 1C).
Lysis by French Press leads to degraded RNAs
A second major lysis method for yeast cells is the French Press. Lysis occurs when the cell suspension is pressed through a small capillary. The pressure difference between the chamber and the capillary ruptures the cell.
Intact RNAs after lysis by mortar grinding
We also tested a third described method for cell lysis, which is grinding deep-frozen cells with a mortar [20–22]. This method breaks cells mechanically while keeping proteins and nucleic acids intact due to the lack of enzymatic activity at ultra-low breaking temperatures. After grinding, the extract is thawed in lysis buffer in the presence of RNase inhibitors (10 mM RVC and 100 U/ml SuperaseIn), immediately inactivating deleterious enzymatic activities.
Both RNase inhibitors are needed to keep mRNA intact
We then checked the effect of a reduced concentration of both RNase inhibitors, SuperaseIn and RVC, on RNA integrity. For this purpose we used a strain where Sub2p, an RNA-binding protein required for splicing and mRNA export, is tagged . Sub2p is more abundant than She2p and believed to bind to mRNAs competent for export . Like She2p, it could also have a protective function against mRNA degradation that could be more prominent due to its higher abundance. We lysed cells by mortar grinding and thawed them in lysis buffer containing either 5 mM RVC and 50 U/ml SuperaseIn, or 5 mM RVC, or 50 U/ml SuperaseIn. We took samples from crude lysate, supernatant of a 100000 × g spin, as well as input and flow through from the IgG immunopurification. As shown in Figure 5B, no degradation can be observed when RNA is stained with methylen blue and the signals from the 25S and 18S ribosomal RNAs are compared. In contrast, degradation of PDA1 mRNA is visible in crude lysates from samples that have been thawed in lysis buffers supplemented only with SuperaseIn as inhibitor (compare signal of RNA from lines 8 and 1 in Figure 5B), in concordance with previous results (Figure 5A). When RVC is present as sole RNase inhibitor (lines 5–7 in Figure 5B and compare with line 1), degradation is reduced. Only when both inhibitors are present in the lysis buffer (lines 2–4 in Figure 5B and compare with line 1) intact RNAs are detected.
Since most RNA-binding proteins fulfil their function in the context of RNA-protein complexes, knowledge of RNAs associated with specific RBPs is essential to elucidate their functions. In order to identify these transcripts, new methods must be developed or existing successful protocols for the identification of protein-protein interactions must be adapted. Although several recent publications have identified RNA partners from RNP-complexes [9, 16], there are so far no reports on the quality of the RNAs purified from these complexes. Here we demonstrate that the method used for cell lysis of yeast cells is of great importance for isolation of intact complexes. Whereas standard lysis methods like disruption by French Press or glass bead mill lead to massive RNA degradation (Figures 1, 2, 3), grinding yeast cells at ultra-low temperatures leaves cellular RNA intact (Figures 4, 5).
The major difference between the lysis methods compared in this study is the timing between addition of the RNase inhibitor and target inactivation. In French Press and glass bead milling, a lag between addition and target inactivation occurs because inhibitors require cells to be broken in order to inactivate RNases as the yeast's cell wall acts as a barrier for large molecules. Since RNases are highly active enzymes, this could result in RNA degradation prior to their inactivation by the inhibitor. This lag could explain the degradation observed in samples from crude lysates produced by these lysis methods even in the presence of RNase inhibitors (Figures 1, 2, 3). This idea is supported by the lack of further degradation during subsequent steps (Figures 2, 3). Although we have only analyzed the first step of the TAP protocol, the IgG immunopurification, the conclusion from this experiment can be extrapolated to immunoprecipitated RNPs as the molecular interactions in both methods are equivalent.
In contrast to bead milling or disruption by French Press, the temperature during grinding is kept close to -80°C and enzymatic activities are essentially absent. The results from Figure 4 show only little RNA degradation after thawing ground extracts in buffer supplemented with RNase inhibitors, which supports cell grinding as an RNA integrity conserving method. Our data demonstrating enhanced stability of mRNAs using the grinding method are supported by previous successful purifications of snRNPs with intact snRNAs using grinding as lysis method [21, 22].
RNA degradation can severely bias identification of RNA from purified RNPs because only RNA fragments that are protected from degradation would remain in the purified extract. Under these circumstances, the choice of the primer for retrotranscribing the pool of purified RNAs is critical since it is likely that only primers annealing close to or inside the protected sites would result in a cDNA product.
Furthermore, when such protected RNA fragments are used to probe micro-arrays, the type of the DNA-array also becomes crucial. Since many RBPs bind to the UTRs of mRNAs and many DNA-microarrays contain only DNA corresponding to the ORF of the genes, detection of bound RNAs by such microarrays could be biased against RBPs binding outside the ORF. This problem would be enhanced using arrays composed of long oligonucleotides since only RNAs whose protected regions overlap with the oligo sequence would be identified.
Another point to consider is the low abundance of several RNPs. As many RNA-binding proteins are expressed at low levels, purification of complexes using RNPs as bait might result in low yield of bait protein plus associated RNAs. Under these circumstances, it is mandatory to keep RNA intact at early stages (in the crude lysate), since the following purification steps might become an additional source of RNA degradation. This idea is also supported by our results (Figure 5A) where, even under optimised lysis conditions, mRNAs become sensitive to degradation during longer incubation times.
Our data suggest that mechanical breakage of frozen yeast cells at ultra-low temperatures is the lysis method of choice for purification of intact mRNP complexes. Since in recent approaches [9, 16] different lysis conditions have been used, a substantial number of protein-associated mRNAs might have been missed.
Strains used for this study. Strains used in this study and its relevant genotype. Modified gene is indicated in bold letters.
MAT a, ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3
MAT a, ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3, NRP1 -TAP::k.l.TRP1
MAT a, ade2-1, trp1-1, can1-100, leu2-3, 112, his3-11, 15, ura3, PBP2 -TAP::k.l.TRP1
MAT alpha, ade2-1, trp1::hisG, can1-100, leu2-3, 112, his3-11, 15, ura3, pep4::LEU2, bar1::hisG, SHE2 -TAP::k.l.TRP1
Oligonucleotides used in this study. Oligonucleotides used in this study for generating the tagged strains.
Yeast cultures were grown at 30°C in YPD media  up to an OD600 nm/ml of 2–3. Cultures were harvested at 4°C and washed with ice-cold sterile water.
Glass bead mill
Cell pellets from 10000 OD600 nm (5 l cultures with an OD600 nm/ml of 2) were harvested, washed with cold sterile water and immediately lysed or the wet pellet snap-frozen in liquid nitrogen. For lysis, harvested cells were resuspended in 30 ml lysis buffer (10 mM K-Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc (Biomol, Germany), 0.7 μg/ml pepstatinA, 100 U/ml SuperaseIn (Ambion, UK), 20 mM Ribonucleoside Vanadyl Complex (RVC, NEB, UK)) and immediately broken. Frozen cells were stored at -80°C until used. Then, they were thawed in 30 ml lysis buffer and immediately lysed. Cells were lysed in a glass bead mill (Bead-beater, Biospec) in 3 cycles (3 min breaking and 5 min cooling). During lysis, cells were kept cold by a water-ice bath.
Cell pellets from 5000 OD600 nm (2.5 l cultures with an OD600 nm/ml of 2) were harvested, washed with cold sterile water and immediately lysed or the wet pellet snap-frozen in liquid nitrogen. For lysis, harvested cells were resuspended in 15 ml lysis buffer (10 mM K-Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA, 100 U/ml SuperaseIn, 20 mM RVC) and immediately broken. Frozen cells were stored at -80°C until used. Then, they were thawed in 25 ml lysis buffer and immediately broken. Lysis was performed as described  with a cold chamber.
Cell pellets corresponding to 3–15 l cultures, with an OD600 nm/ml of 2–3, were frozen by pressing the cell pellet through a 50 ml syringe directly into liquid nitrogen  and stored at -80°C until used. Up to 3 g frozen cells were ground manually in liquid nitrogen. Larger amounts were ground in a motor-driven mortar (Mortar Grinder RM 100, Retsch, Germany) for 15 min at pressure setting 5–7. Mortar and pestle were precooled twice with liquid nitrogen. Crushed dry ice was added continuously during grinding for keeping the mortar cold. Ground material was stored at -80°C until used. Typically, 25 g of ground cells were used for experiments running up to the first TAP immunopurification step, and 1–5 g of ground cells were used for experiments including only differential centrifugation steps. Ground cells were thawed in 1 ml of lysis buffer (40 mM K-Hepes pH 8, 1 mM MgCl2, 0.30% Igepal CA-630, 120 mM NaCl, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA, 200 U/ml SuperaseIn, 40 mM RVC) per gram.
Glass bead mill and French Press
After cell lysis, cell extracts were subjected to serial centrifugations at 4°C. First, the crude lysate (CL) was spun 3 min at 1200 × g to pellet cell debris. The total extract (S1) was further spun for 20 min at 7500 × g and the supernatant collected (S7). Salt and buffer concentration were adjusted to IgG binding conditions (20 mM K-Hepes pH 7.4, 5 mM KCl, 0.75 mM MgCl2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA) with the addition of adjusting buffer (50 mM K-Hepes pH 7.4, 140 mM K-acetate, 30 mM Tris-HCl pH 8, 300 mM NaCl, 3 mM EGTA, 0.3% Igepal CA-630, 1 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 2 μg/ml aprotinin, 1.6 μg/ml bestatin, 2 μg/ml leupeptin, 0.1 mg/ml pefabloc, 1.4 μg/ml pepstatinA). The resulting sample was again spun for 20 min at 20000 × g and the supernatant (S20) finally spun at 200000 × g for 20 min. The supernatant was collected (S200) and the resulting pellet (P200) was resuspended in 10 ml IgG binding buffer (20 mM K-Hepes pH 7.4, 5 mM KCl, 0.75 mM MgCl2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA).
After thawing the ground cells in lysis buffer, the sample was subjected to serial centrifugation at 4°C. First, the crude lysate (CL) was spun 3 min at 1200 × g to pellet cell debris. Total extract (S1) was further spun for 20 min at 7500 × g and the supernatant (S7) was collected. The resulting pellet (P7) was resuspended in 10 ml 0.5 × lysis buffer. Supernatant (S7) was again spun for 20 min at 20000 × g and the supernatant (S20) finally spun at 200000 × g for 20 min. The supernatant was collected (S200) and the resulting pellet (P200) was resuspended in 10 ml 0.5 × lysis buffer.
For TAP purifications of Sub2p, total extract (S1) was directly centrifuged at 100000 × g for 1 h (S100).
The lipid phase was discarded from all fractions.
100–300 μl aliquots from the different fractions were extracted immediately 3–4 times with phenol:chloroform:isoamylalcohol (pH 4), precipitated with sodium acetate and ethanol and stored at -80°C until analyzed.
Glycerol (5% final concentration) was then added to those samples used for further TAP purification steps. These samples were snap-frozen in liquid nitrogen and stored at -80°C for a maximum of 2–3 days. Aliquots were taken before freezing and immediately after thawing to control for degradation during freeze-thawing. These aliquots were extracted, precipitated and stored as described above. TAP purification was performed up to the IgG immunopurification step (first step of the TAP protocol) as described , using as IgG binding buffer 20 mM K-Hepes pH 7.4, 5 mM KCl, 0.75 mM MgCl2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA. Samples were taken before (IgG input) and after IgG immunopurification (flow through, IgG FT) and immediately extracted, precipitated and stored at -80°C as described above.
Total RNA was isolated by direct phenol:chloroform extraction from the same strains used in the study as described , precipitated with sodium acetate and ethanol and kept at -80°C in ethanol until used as quality control. RNA pellets from the different samples were resuspended in DEPC-treated water, ratio A260 nm/A280 nm measured and equivalent amounts of RNA were loaded onto 1.2% agarose-formaldehyde gels. Samples loaded on gels for direct visualization were supplemented with 0.2 μg/μl of ethidium bromide in loading buffer. For Northern blot analysis gels were blotted onto nylon membranes, the membrane stained with methylene blue for determining transfer efficiency and probed against ASH1 and/or PDA1 mRNA (see Figure legend for details).
List of abbreviations
- The abbreviations used are:
tandem affinity purification
ribonucleoside vanadyl complex
RNA recognition motif
open reading frame
tobacco etch virus protease.
This work was supported partially by an individual fellowship to MLdH by Fundación Ramón Areces (Spain) and by research grants to RPJ by the Deutsche Forschungsgemeinschaft (Germany). We want to thank Anja Moraru and Dr. Coral del Val for critical reading of the manuscript and Dr. Katja Strässer (Gene Centre, Munich, Germany) for the Sub2p-TAP strain and for critical reading of the manuscript.
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