Chapter 11 Biochemical Studies of the Mammalian Exosome with Intact Cells

C H A P T E R

E L E V E N

Biochemical Studies of the Mammalian Exosome with Intact Cells Geurt Schilders and Ger J. M. Pruijn Contents 212

1. Introduction 2. Identifying Protein–Protein Interactions by the Mammalian Two-Hybrid System 2.1. Protocol 2.2. Comments 3. Characterization of Different Exosome Subsets by Glycerol Sedimentation 3.1. Protocol 3.2. Comments 4. Studying Exosome Function with RNAi 4.1. Protocol 4.2. Comments References

213 214 216 218 219 220 222 222 223 224

Abstract A key component responsible for 30 - to 50 -RNA turnover in eukaryotic cells is the exosome, a multisubunit complex present in both the nucleus and cytoplasm of the cell. Here we describe several methods that can be applied to study the structure and function of the exosome in mammalian cell lines. The mammalian two-hybrid system has been successfully used to identify protein–protein interactions between exosome core components. Cell and glycerol gradient fractionation procedures are described that allow the identification and characterization of different exosome subsets. Finally, a protocol to study the function of the exosome in RNA turnover with RNA interference is presented.

Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands Methods in Enzymology, Volume 448 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)02611-6

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2008 Elsevier Inc. All rights reserved.

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1. Introduction The exosome is a multisubunit complex containing 30 - to 50 -exoribonuclease activity and has been shown to be vital for several cellular processes, such as mRNA turnover (Bousquet-Antonelli et al., 2000; Das et al., 2003; Jacobs Anderson et al., 1998; van Dijk et al., 2007), ribosomal and small nucle(ol)ar RNA processing (Allmang et al., 1999a; Schilders et al., 2005; Stoecklin et al., 2005; van-Hoof et al., 2000) as well as several RNA surveillance pathways (Houseley et al., 2006; Kadaba et al., 2006; LaCava et al., 2005; Vanacova et al., 2005; Wyers et al., 2005). The exosome and its associated proteins can be divided into several types of subunits. The human exosome contains nine components that are shared by the cytoplasmic and nuclear complex and are, therefore, termed ‘‘core’’ components (Chen et al., 2001). Accessory components are defined as active 30 - to 50 -exoribonucleases, which contribute to the catalytic activity of the core exosome. Examples are PM/ Scl-100 (Rrp6p in yeast), which belongs to the RNase D family of exoribonucleases, and Dis3p/Rrp44p, a member of the RNase R family of exoribonucleases, which so far has been found to be associated with the exosome in several eukaryotes, but not in humans. Auxiliary proteins can be defined as proteins that either directly or indirectly (e.g., via accessory proteins) interact with the core complex and aid in the function of the exosome, such as RNA helicases and RNA-binding proteins. Six of the human core components (hRrp41, hRrp42, hMtr3, hRrp46, PM/Scl-75 [Rrp45 in yeast], and OIP2 [Rrp43 in yeast]) contain an RNase PH domain (RPD), whereas the other three components (hCsl4, hRrp4, hRrp40) contain an S1 domain in addition to a KH or zinc-ribbon domain. The assembly of the complex formed by the nine core components was found to be similar to the eubacterial polynucleotide phosphorylase (PNPase) complex, suggesting that their three-dimensional structure might also be similar. The crystal structure of PNPase revealed that the S1 domains form a crown of RNA-binding domains around the space in the center of the trimer (Symmons et al., 2000). On the basis of the PNPase structure and taking into account the mutual subunit interactions observed in different two-hybrid systems, a model for the human exosome was generated in which the RNase PH domain containing subunits form a hexameric ring, and the three proteins with the S1 (and KH or zinc-ribbon) RNA binding domains are positioned at the outer surface of this ring (Lehner et al., 2004; Raijmakers et al., 2002). Biophysical support for this ringlike structure was provided by the crystal structure of the reconstituted human exosome. The structure that revealed that the arrangement of the exosome components is consistent with the model of the human exosome, although the S1 domain proteins were found to be associated with one side

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of the ring rather than with the periphery of the ring (Liu et al., 2006) (see also Chapter 10 by Greimann and Lima in this volume). Nevertheless, these data showed that it is possible to derive a reliable model of a multisubunit complex from the results obtained in a mammalian two-hybrid system. In yeast, the exosome was reported to be present in differently sized complexes (Mitchell et al., 1997). Also, in humans, the core exosome has been reported to be associated with different complexes sedimenting at 10S and 60 to 80S in glycerol gradients (Schilders et al., 2005; van Dijk et al., 2007). For the U3 snoRNP, the high-molecular-weight (60 to 80S) complexes were shown to represent preribosomes (Granneman et al., 2004; Lukowiak et al., 2000). Therefore, the sedimentation of the human exosome in similarly sized complexes suggested that the 60 to 80S complexes correspond to exosomes associated with preribosomal complexes. Interestingly, a subset of exosome-auxiliary proteins have been identified that only cosediment with the 60 to 80S complexes and not with the 10S complexes, suggesting that these proteins are involved in ribosome synthesis. Indeed, both the human exosome and the exosome-auxiliary proteins cosedimenting with the 60 to 80S exosome complexes have been implicated in the 30 -end processing of the 5.8S rRNA (Schilders et al., 2005; Stoecklin et al., 2005). These data demonstrated that by biochemical fractionation experiments, different exosome subsets containing additional auxiliary proteins could be identified and characterized. Most of the biochemical and genetic approaches that have been applied in the past to identify the functions of the exosome and its accessory and auxiliary components have used the yeast Saccharomyces cerevisiae or in vitro systems with individual components. With the possibility of RNA-interference (RNAi) technology in mammalian cells (Elbashir et al., 2001), the study of exosome function has been extended to identify the role of the mammalian exosome in rRNA processing (Schilders et al., 2005; Stoecklin et al., 2005), mRNA turnover (Stoecklin et al., 2005; van Dijk et al., 2007), and mRNA surveillance pathways, such as for mRNAs containing a premature translation termination codon (Lejeune et al., 2003). It is anticipated that RNAi will probably also be applied in combination with the microarray technology to identify the role of the exosome in the expression of protein coding genes and small noncoding RNAs (sn(o)RNAs, miRNAs) on a genome-wide scale. Here we describe several protocols that have been successfully applied in our laboratory to study the human exosome.

2. Identifying Protein–Protein Interactions by the Mammalian Two-Hybrid System One of the major advantages of studying the interaction between two mammalian proteins in the mammalian two-hybrid system is that these proteins are expressed in their ‘‘natural’’ environment (i.e., the most optimal

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conditions for proper folding, posttranslational modification, and oligomerization). For studying protein–protein interactions with the Checkmate mammalian two-hybrid system (Promega), three plasmids are cotransfected into mammalian cells. The pACT plasmid contains a VP16 transcriptional activation domain upstream of the cDNA of interest, whereas the pBIND plasmid contains a GAL4 DNA-binding domain sequence upstream of the second cDNA of interest. The third pG5luc plasmid contains five GAL4 binding sites upstream of a firefly luciferase gene that acts as a reporter for the interaction between the proteins to be studied (Fig. 11.1). It should be noted that the pBIND plasmid also expresses the Renilla luciferase, which allows for normalization of differences in transfection efficiency. The detailed protocol described in the following is optimized for COS-1 cells and FuGENE transfection reagent. However, other mammalian cell lines and transfection reagents can be used as well, although this may require minor optimization.

2.1. Protocol 1. 3 to 4  105 actively growing COS-1 cells are seeded into a single well of a 6-well plate (Greiner Bio-One) and incubated overnight in 2 ml culture medium (DMEM containing 10% heat-inactivated fetal calf serum [FCS]) at 37  C in a CO2 incubator. 2. Per transfection 1 mg of pACT and pBIND, either with or without insert, and 1 mg of the pG5luc reporter plasmid (Promega) are added to 100 ml of DMEM. 3. To the DNA/DMEM mixture, 4 ml of FuGENE (Roche) is added and, after gentle mixing, incubated for 20 min at room temperature. 4. Meanwhile, the COS-1 cells are washed twice with PBS and incubated with 4 ml DMEM containing 10% FCS. 5. The FuGENE/DNA complexes are added to the cells and gently mixed. 6. After incubation for 48 h at 37  C in a CO2 incubator, cells are washed twice with PBS and lysed by the addition of 500 ml of passive lysis buffer (Dual Luciferase Reporter (DLR)-kit [Promega]). 7. After lysing the cells for 20 min on a shaking incubator at room temperature, cell extracts are transferred to a tube and placed on ice. 8. The expression levels of both the firefly luciferase and the control Renilla luciferase are determined by activity measurements with the Dual Luciferase Reporter Assay System (Promega) and a Lumat LB 9507 Luminometer (Berthold). 9. For the firefly luciferase activity, 100 ml of firefly luciferase substrate is added to 20 ml of cell extract, and the luminescence is measured to detect the interaction between the proteins of interest.

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Protein2 GAL4 B S

V GAL CM

pBIND

pG5 luc

M

V

pACT

Firefly

Sv 4 0

NLS VP16

Protein1

C

Renilla

Co-transfect into COS-1

Prot. 2

Prot. 1 VP16

GAL4 GAL4 GAL4 GAL4 GAL4 GAL4

Firefly luciferase

Prot. Prot. 2 1 GAL4

VP16

GAL4 GAL4 GAL4 GAL4 GAL4

Firefly luciferase

Figure 11.1 Principle of the checkmate mammalian two-hybrid system. The pACT, pBIND, and pG5luc vectors are introduced into COS-1 cells with FuGENE. After culturing for 48 h, cells are lysed and extracts analyzed for luciferase activity. The pG5luc vector contains five GAL4 binding sites upstream of the firefly luciferase gene. When the fusion partners of the GAL4 DNA-binding domain (from pBIND) and the VP16 activation domain (from pACT) do not interact, luciferase expression will not be induced, resulting in little firefly luciferase activity. An interaction between the two fusion proteins on the other hand will result in an increase of firefly luciferase expression.

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10. To monitor transfection efficiency, Renilla luciferase activity is measured by the addition of 100 ml of Renilla luciferase substrate solution to the mixture of step 8, and luminescence is measured. 11. After obtaining the raw data, the following calculations can be performed. The observed firefly luciferase activity can be normalized for differences in the transfection efficiency by dividing the firefly luciferase activity values by the Renilla luciferase activity values observed with the same cell extract and multiplied by 1000. Subsequently, the luciferase activity can be expressed in relative luminescence units (RLU). In all experiments we included hRrp42 in pBIND and hCsl4 in pACT as a positive control and set the normalized luminescence resulting from the interaction between these two exosome subunits at 100 RLU. This approach allowed a direct comparison of the results obtained in the distinct experiments. In addition, it is crucial to include background controls in every experiment (i.e., luciferase measurements with extracts from cells transfected with either the ‘‘empty’’ pACT or the ‘‘empty’’ pBIND vector, which replaces the cDNA containing plasmid). An example of a mammalian two-hybrid experiment is shown in Table 11.1.

2.2. Comments It is important to note that the two-hybrid system can result in false-positive interactions, which might result from activation of the reporter independent of the protein interaction or because of (weak) nonspecific binding. Therefore, it is important to determine a cutoff value. In the two-hybrid experiments we have carried out, an interaction is considered positive when the RLU exceeds the sum of the corresponding ‘‘empty-vector’’ control measurements. For the experiment documented in Table 11.1, this results in the sum of 48 + 45 RLU, which is well below the observed 292 RLU for the interaction of C1D with PM/Scl-100. Note that false-negative results can also occur. Several common problems could be: the protein is not properly modified; the protein is not expressed to sufficiently high levels; the protein is insoluble; fusion of the protein to the GAL4 or VP16 domain interferes with the proper folding of the protein or shields the interaction surface. Expression levels can easily be tested by Western blot analysis with specific antibodies directed against the GAL4 and VP16 domains. If Western blot analysis shows that a protein is poorly expressed, expression can be enhanced by the addition of sodium butyrate, which is known to stimulate expression from the CMV promoter (Wilkinson et al., 1992). If sodium butyrate is to be used, it should be added 24 h after transfection to a final concentration of 5 mM. Another factor that may lead to false-negative results is the subcellular localization of the fusion protein. The interaction between the proteins of interest has to occur in the nucleus to induce transcription of the firefly

Table 11.1 Example of a single two-hybrid interaction between C1D and PM/Scl-100

a b

Plasmids

Firefly luciferase activitya

Renilla luciferase activitya

pACT-hCsl4 pBIND-hRrp42

26135

3308947

pACT-empty pBIND-C1D

15745

4085106

pACT-PM/Scl-100 pBIND-empty

4354

1229814

pACT-PM/Scl-100 pBIND-C1Db

47934

2079513

Arbitrary units. Interaction should be corrected for background levels: 292  48  45 = 199.

Normalized luciferase activity

(26135/ 3308947)  1000 = 7.90 (15745/ 4085106) 1000 = 3.85 (4354/1229814)  1000 = 3.54 (47934/ 2079513)  1000 = 23.05

Relative luminescence units (RLU)

7.90  (100/7.90) = 100 3.85  (100 / 7.90) = 48 3.54  (100/7.90) = 45 23.05  (100/7.90) = 292

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luciferase gene. The subcellular localization can be tested by indirect immunofluorescence and by use of the antibodies directed to the GAL4 or VP16 domains. Note that the pACT vector contains a nuclear localization sequence (NLS) in front of the VP16 fusion protein that might circumvent the problem in most cases. In contrast the pBIND vector does not contain a NLS, but presumably the proteins will already interact in the cytoplasm and may be exported into the nucleus as a heterodimer by means of the NLS present in the pACT vector.

3. Characterization of Different Exosome Subsets by Glycerol Sedimentation As described previously, the human exosome is composed of a core consisting of nine subunits conserved in both the nuclear and cytoplasmic exosomes, as well as auxiliary proteins that are only associated with a subset of exosome complexes. To identify different exosome-containing macromolecular complexes, biochemical fractionation experiments can be performed in which total or compartment-specific cell extracts can be fractionated by glycerol gradient sedimentation (Fig. 11.2). Subsequently, distribution of the exosome complexes in the gradient can be monitored by Cell extract

Low glycerol concentration

5%

5%

18 h, 100 000 ⫻ g

Fractions 5%

High glycerol concentration

Glycerol

Western blot analysis

40%

40%

5% 12 S

40%

40 S

40% Glycerol Markers

60 S

Anti-hRrp4 i

1

5

10

15

20

23

Fractions

Figure 11.2 Schematic representation of glycerol gradient sedimentation analysis. A HEp-2 cell lysate is loaded onto a 5 to 40% glycerol gradient and after centrifugation for 18 h at 100,000g, fractions are collected manually, separated with SDS-PAGE, and analyzed by Western blot analysis. The sedimentation of hRrp4 is visualized by immunoblotting with a mouse monoclonal anti-hRrp4 antibody.

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Western blot and immunoprecipitation analysis. In addition, activity assays can be performed with either the gradient fractions or the immunoprecipitated material to functionally characterize the exosome subsets. In the following section a method is described for the fractionation and characterization of different exosome subsets in mammalian cells.

3.1. Protocol 3.1.1. Preparation of cytoplasmic and nuclear extracts with digitonin 1. 5  106 HEp-2 cells are grown to 70% confluence, harvested, and resuspended in 500 ml lysis buffer (20 mM HEPES/KOH at pH 7.6, 150 mM NaCl, 0.5 mM DTE , 0.5 mM PMSF). 2. Permeabilization of the plasma membrane is achieved by the addition of digitonin to a final concentration of 0.025%. After mixing gently, the cells are incubated for 10 min at room temperature. Note that lysis by digitonin is selective for cholesterol-containing membranes like the plasma membrane, with minimal nuclear permeabilization and leakage of nuclear proteins. 3. The lysate is centrifuged at 1000g for 5 min at room temperature. 4. The supernatant, which contains the cytoplasmic material, is transferred to a new tube. 5. The pellet is resuspended in 500 ml lysis buffer and is used to prepare the nuclear extract. 6. Both the cytoplasmic and the nuclear material are homogenized by sonication for 3  20 sec with a Branson microtip. 7. Triton X-100 is added to the homogenates to a final concentration of 0.2% (v/v). 8. Lastly, insoluble material is removed by centrifugation at 12,000g for 10 min at 4  C. The purity of the nuclear and cytoplasmic fractions can be determined by Western blot analysis. As a nuclear marker, we use a polyclonal serum (from an autoimmune patient) that recognizes topoisomerase I, whereas a mouse monoclonal antibody directed against eIF2a is used as a cytoplasmic marker. It is particularly important to check for leakage of proteins from the nucleus during cell permeabilization, and a relatively soluble nuclear protein should be used to monitor proper cell fractionation, because proteins stably associated with chromatin, the nucleolus, or other higher order nuclear substructures will not easily leak from a permeabilized nucleus. 3.1.2. Preparation of 5 to 40% glycerol gradients 9. For each gradient, two times 8 ml of lysis buffer supplemented with 0.2% Triton X-100 is prepared containing 5 or 40% glycerol, respectively.

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10. Add 6 ml of 5% glycerol lysis buffer to the bottom of an ultracentrifuge tube (the tube should be compatible with the rotor required for the centrifugation step) (see step 6). 11. Subsequently, 6 ml of 40% glycerol lysis buffer is added slowly to the bottom of the tube with a long needle, while not disturbing the 5% glycerol layer. Note that the order of steps is important for creating an optimal gradient. 12. The 5 to 40% glycerol gradient is generated with a Biocomp Gradient Master 107. Note that depending on the size of the complexes to be analyzed and the resolution of complexes in a particular region of the gradient, glycerol gradients with different glycerol concentration ranges can also be used. 13. The cell lysate (0.5 ml), prepared as described previously, is layered on top of the 12-ml gradient. 14. The gradients are centrifuged in a Sorvall TH641 (or equivalent swingout) rotor for 18 h at 100,000g at 4  C. 15. After centrifugation, 500-ml fractions are collected manually by a micropipet from top to bottom and stored at 20  C until further analysis.

3.2. Comments The collected fractions can be separated with SDS-PAGE, and the proteins of interest can be visualized by Western blotting. Note that when the pellet fraction from the gradient is also analyzed, it can contain large protein aggregates resulting in a false-negative signal in this fraction. An overview of available anti-exosome subunit antibodies is given in Table 11.2. In our experiments, core exosome components seemed to be slightly more abundant in the nuclear fraction of HEp-2 cells (Brouwer et al., 2001). In contrast to yeast, where Rrp6p is restricted to the nucleus, PM/Scl-100 is found in substantial amounts in the cytoplasm, whereas the auxiliary proteins MPP6 and C1D are restricted to the nuclear fraction. With regard to the distribution of exosome complexes in glycerol gradients, the nuclear exosome seems to be equally divided between high-molecularweight fractions and low-molecular-weight exosome complexes. The latter most likely represent core exosomes associated with PM/Scl-100. In the cytoplasm, most exosome complexes are found as low-molecular-weight complexes, and only a small percentage seems to be stably associated with larger complexes (van Dijk et al., 2007). RNA can be isolated from the gradient fractions with TRIzol (Invitrogen) and analyzed with denaturing gel electrophoresis followed by Northern blot hybridization. As markers for 40S and 60S complexes, fractionation of the 18S and 28S rRNAs can be monitored with agarose gel electrophoresis and ethidium bromide staining. As an additional marker

Table 11.2 Antibodies and siRNAs to study the mammalian (human) exosome Protein

Antibody

siRNA (sequence of sense strand (50 to 30))

Reference(s)

hRrp4

AGCUUUCACACAGAUCAACdTdT

(Allmang et al., 1999b; Schilders et al., 2005; van Dijk et al., 2007)

hRrp40

Rabbit polyclonal Mouse monoclonal Rabbit polyclonal

GAAUAUGGGUUAAGGCAAA

OIP2 PM/Scl-75

Rabbit polyclonal Rabbit polyclonal

hRrp41

Rabbit polyclonal

hRrp46

Rabbit polyclonal

PM/Scl-100

Rabbit polyclonal

hMtr4 C1D MPP6

— — Rabbit polyclonal

(Brouwer et al., 2001; Stoecklin et al., 2005) ( Jiang et al., 2002) (Mukherjee et al., 2002; Stoecklin et al., 2005; van Dijk et al., 2007) (Brouwer et al., 2001; Lejeune et al., 2003; Schilders et al., 2005) (Brouwer et al., 2001; Stoecklin et al., 2005) (Brouwer et al., 2001; Lejeune et al., 2003) (Schilders et al., 2007) (Schilders et al., 2007) (Schilders et al., 2005)

hSki3 hSki8

Rabbit polyclonal Rabbit polyclonal

— GCGUGAUCCUGUACCAUUA GCCAAGAUGCUCCCAUAAUdTdT UGUGCAGGUGCUACAGGCAdTdT GCAAAGAGAUUUUCAACAA CAACACGUCUUCCGUUUCU GUACAACCCAGGAUAUGUGdTdT GCCUAUGCACUUCAAAUGAdTdT UUGUUCAAGUGGAUCCAACdTdT GAGCACUGGUACUUGGAUUdTdT CAGUAGAGCUUGAUGUGUCdTdT GAUAUGAGACCUUGGUGGGdTdT — UGACCAACCAGUACGGUAUdTdT

(Zhu et al., 2005) (Zhu et al., 2005)

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for 12S and 60 to 80S complexes in the gradient, a U3 snoRNA probe can be used. Immunoprecipitations can be performed directly with the collected fractions, and the precipitated complexes can be further characterized. For example, the activity of the complex can be determined with an in vitro exonuclease activity assay (Brouwer et al., 2001; Mitchell et al., 1997).

4. Studying Exosome Function with RNAi The identification of small interfering RNAs (siRNAs) that are able to suppress the expression of genes in a sequence-specific manner in cultured mammalian cells has made it possible to study the function of the exosome in human cells and has revealed that the exosome plays a key role in several 30 - to 50 -RNA turnover pathways. Here, we describe a general procedure for the downregulation of exosome components with RNAi (RNAi) in human cell lines.

4.1. Protocol 1. HEp-2 cells, grown to 70% confluent monolayers are seeded in a 6-well plate (approximately 1.5  105 cells/well) and cultured overnight at 37  C in a humidified 5% CO2 incubator. 2. For each well in a 6-well plate, 2 ml of a 20 mM siRNA stock solution is diluted in 183 ml OptiMEM (Gibco) and gently mixed. 3. 3 ml of Oligofectamine (Invitrogen) is diluted in OptiMEM to a final volume of 15 ml and after gentle mixing incubated for 10 min at room temperature. 4. After the 10-min incubation, the diluted Oligofectamine is added to the diluted siRNA, gently mixed, and incubated for 20 min at room temperature. 5. Meanwhile, the HEp-2 cells are washed twice with PBS and incubated with 800 ml of DMEM (+10% FCS). For most cell lines it is best to use serum-free culture medium during transfection, although for HEp2 cells, FCS containing medium can be used without loss of transfection efficiency. 6. The 200 ml of siRNA-Oligofectamine mixture is added to each well, and the 6-well plate is gently swirled. Note that it is important to mix the transfection components just before its addition to the cells to achieve a high and reproducible transfection efficiency. 7. The cells are incubated for 4 h at 37  C in a 5% CO2 incubator. 8. Two ml of DMEM containing 10% FCS is added.

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9. If the cells are cultured for periods longer than 48 h, it is necessary to retransfect the cells, because we have observed a reduction in knockdown efficiency 48 h post-transfection.

4.2. Comments The optimal incubation times, amount of siRNA, and transfection reagents should be determined empirically for each cell type. However, the protocol described above should be a good starting point for optimization for most mammalian cell lines, for example, by replacing Oligofectamine with an equal amount of an alternate transfection reagent. With regard to efficient and successful exosome knockdown experiments, there are some specific points to bear in mind. We observed that knockdown of core exosome components, such as hRrp41 and hRrp4, by RNAi is more efficient in the cytoplasm than in the nucleus (Fig. 11.3). Therefore, RNAi effects observed until 48 h after transfection are likely to be due primarily to knockdown of the cytoplasmic exosome. For studying the function of core exosome components in the nucleus, it will be required to elongate the transfection time to at least 72 h. However, it should be clear that the effects observed at that time point might as well be indirectly caused by the impairment of cytoplasmic exosome function. Moreover, knockdown of core exosome components inhibits cell growth, which could lead

1

2

3

4

1

2

3

4

hRrp41 PM/Scl-75 PM/Scl-100 Cytoplasm 1 Control siRNA

3 hRrp41 siRNA

2 PM/Scl-75 siRNA

4 PM/Scl-100 siRNA

Nucleus

Figure 11.3 Exosome subunit knockdown efficiency in different cellular compartments. Knockdown of hRrp41, PM/Scl-75, and PM/Scl-100 by RNAi was analyzed with Western blotting and nuclear and cytoplasmic extracts prepared from HEp-2 cells. In the cytoplasmic fractions, significantly reduced levels of hRrp41, PM/Scl-75, and PM/ Scl-100 were observed, whereas in the nuclear fractions only an efficient knockdown of PM/Scl-100 was detected. Importantly, this demonstrates that the cytoplasmic exosome is more efficiently downregulated than the nuclear exosome (see also Van Dijk et al. [2007]).

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to nonspecific side effects. PM/Scl-100 on the other hand is efficiently downregulated in the nucleus and, thus, might be a more attractive target to downregulate the function of the nuclear exosome (see Fig. 11.3). If reporter constructs are used, such as b-globin mRNA expression constructs to study 30 - to 50 -mRNA turnover on exosome knockdown, approximately 8  106 HEp-2 cells are grown to 70% confluent monolayers and are transfected by electroporation with 20 mg of plasmid DNA in 900 ml of DMEM containing 10% FCS. HEp-2 cells are electroporated at 260 V and 950 mF with a Gene-Pulser II (Bio-Rad), subsequently seeded in a 6well plate (approximately 1.5  105 cells/well), and cultured overnight at 37  C in a humidified 5% CO2 incubator. Then the siRNA transfection protocol just described can be applied to study the effects on the expression of the reporter mRNA. Note that it is important to discriminate between the effects of exosome depletion on mRNA degradation and on other processes that influence steady-state mRNA levels, such as transcription. To do so, we use HEp-2 cells stably transfected with the plasmid pTettTAk, which expresses a transcriptional activator in the absence of tetracycline. The addition of tetracycline results in the inhibition of transcription of the reporter constructs, which eliminates the possibility that the effects are due to transcriptional events. It has been reported for the Trypanosoma brucei exosome that knockdown of a single core exosome component results in codepletion of other exosome components (Estevez et al., 2003). Similarly, it should be noted that knockdown of the human core exosome components hRrp41 and hRrp4 by RNAi also leads to destabilization of other exosome components, which makes it difficult to study the function of a single exosome component by this technology. On the basis of the crystal structure of the human exosome, it is expected that knockdown of other exosome components will also result in the codepletion of the other core exosome components, which makes RNAi unsuitable to study the role of an individual exosome component. However, PM/Scl-75 seems to be an exception, because no codepletion of other core components is observed upon knockdown of PM/Scl-75 (Van Dijk et al., 2007). Also PM/Scl-100 can be downregulated without reducing the levels of core subunits. SiRNAs that have been successfully applied to downregulate the levels of exosome subunits in human cells are also listed in Table 11.2.

REFERENCES Allmang, C., Kufel, J., Chanfreau, G., Mitchell, P., Petfalski, E., and Tollervey, D. (1999a). Functions of the exosome in rRNA, snoRNA and snRNA synthesis. EMBO J. 18, 5399–5410. Allmang, C., Petfalski, E., Podtelejnikov, A., Mann, M., Tollervey, D., and Mitchell, P. (1999b). The yeast exosome and human PM-Scl are related complexes of 30 - to 50 exonucleases. Genes Dev. 13, 2148–2158.

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