Rab1b Silencing Using Small Interfering RNA for Analysis of Disease‐Specific Function

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Rab1b Silencing Using Small Interfering RNA for Analysis of Disease-Specific Function Darren M. Hutt* and William E. Balch*,† Contents 2 4 4 5 6 6 8 9

1. Introduction 2. Materials for siRNA Silencing of Rab1b 3. Procedure for Transfection of siRNA 4. Titration of Optimal siRNA Concentration 5. SDS-PAGE Analysis of Rab1 Knockdown 6. SDS-PAGE Analysis of CFTR 7. Discussion References

Abstract Rab1 GTPase is a critical component required for endoplasmic reticulum (ER)to-Golgi as well as intra-Golgi trafficking. It is required for the proper recruitment of tethering factors to mediate vesicle docking and subsequent fusion to the target membrane compartment. Much is known about the role of Rab1 in ER-to-Golgi trafficking through overexpression of dominant negative mutation that inhibit GTP binding or GTPase activity of the protein, as well as through the use of antibodies to inhibit endogenous protein activity. These techniques have allowed for the establishment of a central role for Rab1 in trafficking in the early secretory pathway; however, the use of these techniques is limited. The introduction of antibodies relies on permeabilization of the cell membrane for their introduction. The use of dominant negative mutations relies on the mutation overwhelming the endogenous protein activity without removing it from the cell. The advent of siRNA to silence genes of interest provides a means to overcome this limitation. Here we describe optimal conditions for the efficient silencing of Rab1b using siRNA to analyze its role in disease.

* {

Department of Cell Biology, The Scripps Research Institute, La Jolla, California The Institute for Childhood and Neglected Diseases, The Scripps Research Institute, La Jolla, California

Methods in Enzymology, Volume 438 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)38001-4

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2008 Elsevier Inc. All rights reserved.

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1. Introduction With over 70 mammalian isoforms, Rab GTPases represent the largest of the five subfamilies of the Ras superfamily of proteins (Pereira-Leal and Seabra, 2001) with each isoform thought to mediate a specific trafficking step. Rab GTPases, as their Ras counterparts, cycle between an active membrane–associated, GTP-bound, and inactive cytosolic, GDP-bound states, and are therefore ideal molecular switches for regulating the orderly progression of vesicles through membranous compartments of the eukaryotic exocytic and endocytic pathways. Following activation by the guanine nucleotide exchange factor (GEF), Rab GTPases mediate the recruitment of effector proteins, which include tethering and fusion factors, to mediate the docking and fusion of trafficking vesicles with its targeted acceptor membrane compartment (Gurkan et al., 2005; Pfeffer and Aivazian, 2004). Following membrane docking and fusion, a GTPase-activating protein (GAP) initiates the GTP hydrolysis of Rab proteins, resulting in inactivation of the protein as well as the generation of a binding target for the GDP dissociation inhibitor (GDI). The latter mediates the extraction of Rab GTPases from the acceptor membrane compartment and directs it’s recycling to the donor compartment. Rab1 had been reported to localize to both the endoplasmic reticulum (ER) and the Golgi compartments (Plutner et al., 1991), suggesting that it might mediate the trafficking between these compartments. In vitro reconstitution experiments in yeast indicate that the docking of ER-derived vesicles is sensitive to the GTPase, Ypt1p, which in turn regulates Uso1pdependent tethering of donor vesicles to a target membrane (Cao et al., 1998). This suggests that Ypt1p mediates tethering of ER-derived vesicles prior to membrane fusion. This observation is consistent with the proposed role of Rab1, the mammalian ortholog of Ypt1p, in mediating the docking of ER-derived vesicle with the cis-Golgi compartment. Rab1 has been shown to recruit the cytosolic tethering factors p115 to the vesicle (Allan et al., 2000) and to interact with the Golgi membrane proteins GM130 and GRASP (Moyer et al., 2001) to mediate the docking process. In addition, antibodies specific for Rab1 were able to block ER to Golgi trafficking as well as intra-Golgi trafficking in semipermeable cells (Plutner et al., 1991). These data support a critical role for Rab1 in ER to Golgi trafficking. In order to characterize the role of Rab1 in trafficking various cargo proteins through the endomembrane compartments of the cell, we have relied on the availability of dominant negative mutations and antibodies to inhibit the function of this protein. The use of these tools is somewhat limited, namely, that the overexpression of mutant Rab1 is dependent on

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efficiency of transfection in the cell line of interest as well as the ability of the given mutant to overcome the endogenous Rab1 and act as a dominant negative. In addition, the introduction of antibodies into the cytosol of cells has proven to be a useful tool to block endogenous protein function. However, this invasive technique requires microinjection or the permeabilization of the plasma membrane, which renders the given experiment into an artificial system since the normal homeostasis of the cell is compromised. In recent years, the use of small inhibitory RNA (siRNA) molecules as a silencing tool has grown exponentially. This technique calls for the introduction of a short RNA sequence, typically 19 nucleotides in length, which is directed to a sequence-specific region in a target gene without off-target silencing of other potentially critical genes. The siRNA is introduced using various techniques, including cationic lipids, which is reminiscent of standard transfection techniques used for the introduction of plasmid DNA. There is now an extensive literature and a large number of companies that design siRNA probes that have minimal ‘‘off-target’’ (e.g., nonspecific) effects that can be used for silencing studies. In order to ascertain that Rab1b knockdown resulted not only in decreased levels of endogenous Rab1, but also in impaired ER to Golgi trafficking, we monitored the maturation of the cystic fibrosis transmembrane conductance regulator (CFTR), the gene mutated in cystic fibrosis (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989). The gene codes for a 1480–amino-acid chloride channel that localizes to the apical plasma membrane of epithelial cells (Anderson et al., 1991a,b). As CFTR is processed in the ER, it acquires its N-linked core glycosylation. This glycosylation is further processed to an endoglycosidase H-resistant form as it matures in the Golgi compartment. These two glycoforms of the protein migrate at 135 and 160 kDa, respectively, on SDS-PAGE, and are referred to as band B and C, respectively. This differential migration of the ER and Golgi forms of the protein allows for a direct measure of the trafficking and maturation of CFTR by Western blotting (Cheng et al., 1990). Although there are currently more than 1500 mutations linked to cystic fibrosis, greater than 90% of patients have at least one copy of the CFTR gene containing a three–base-pair deletion resulting in the loss of phenylalanine at position 508 (DF508). This mutation results in a trafficking-deficient form of CFTR that is restricted to the ER, and therefore is only detectable on SDS-PAGE as the band B glycoform (Cheng et al., 1990). Herein, we describe the methodologies required for the efficient silencing of Rab1b in a lung cell line and an analysis of its effect on the trafficking of both the wildtype CFTR and DF508 CFTR.

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2. Materials for siRNA Silencing of Rab1b Purified siRNA duplex–targeting human Rab1b was purchased from Ambion (cat. no. AM16104), and the sense strand sequence was GAUCCGAACCAUCGAGCUGtt. The nontargeting siRNA sense strand sequence used for these experiments was GCGCGCUUUGUAGGAUUCtt and was purchased from Dharmacon. HiPerFect transfection reagent was purchased from Qiagen. Rab1 was detected using a previously characterized polyclonal antibody (p68) (Plutner et al., 1991), and CFTR was detected using the M3A7 ascites raised against the nucleotide-binding domain 2 (NBD2) of human CFTR, which was obtained from J. R. Riordan, PhD (University of North Carolina). Human bronchial epithelial cells (HBEs) and cystic fibrosis bronchial epithelial cells (CFBE41o-) were cultured in growth media: a-MEM culture media (Gibco cat. no. 12000–063) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 10% FBS, 2 mM L-glutamine, and 1 mg/ml blasticidin or 2 mg/ml puromycin, respectively.

3. Procedure for Transfection of siRNA 1. One day prior to transfection HBE or CFBE, cells are seeded at a density of 1.0
105 cells per well of a 12-well dish, and cultured overnight in growth media. This cell density will allow cells to be confluent 3 days post-transfection. 2. On the day of transfection, remove the growth media and replace with 1.1 ml of fresh growth media. 3. Dilute the siRNA to a 12
working concentration in 100 ml of a-MEM without serum and add 6 ml of HiPerFect reagent. Vortex the mixture at 75% maximum for 5 s. This mixture is for each well of a 12-well culture dish that is to be transfected. 4. Incubate the mixture for 10 min at room temperature to generate transfection complexes. 5. Add the mixture drop-wise to the well containing 1.1 ml of growth media, and incubate for 48 h at 37 /5% CO2. 6. Following the initial 48-h incubation, replace the growth media containing transfection complexes with 1.1 ml of fresh growth media. 7. Prepare transfection complexes as in steps 3 to 5. 8. Culture cells for an additional 48 h. 9. Cells are harvested following 96 h for analysis.

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4. Titration of Optimal siRNA Concentration In order to avoid transfection-related toxicity, it is necessary to optimize the siRNA concentration for efficient silencing of Rab1b. HBE cells were transfected with a complex composed of 6 ml of HiPerFect transfection reagent and 5, 15, or 50 nM final concentration of Rab1b siRNA or 50 nM final concentration of the scrambled control siRNA as described above. One set of cells was transfected once with the indicated siRNA concentration and cultured for 2 days post-transfection, while the second set of cells was transfected twice with the indicated concentration of siRNA at 2-day intervals and cultured for 2 days after the final transfection as described above (Fig. 1.1). These results show that cells which were only transfected with a single hit of siRNA only had a maximal knockdown of Rab1 of 20.8  0.8% at the highest concentration of siRNA (50 nM) A

50 nM Scrambled A B C

5 nM Rab1b A

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15 nM Rab1b A

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40 10 20 30 Concentration siRNA (nM)

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Figure 1.1 Titration of siRNA for Rab1b knockdown. (A) HBE cells were transfected with the indicated siRNA concentration for one hit (upper panel) or two hits (lower panel) and cultured for 2 days post-transfection. Rab1 was detected on Western blot using the polyclonal antibody, p68. (B) Quantitative analysis of Rab1 knockdown in HBE cells.The amount of Rab1 in HBE cells following transfection with 5, 15, or 50nM siRNA for one hit (solid squares) or two hits (open squares) is quantified as a percent of Rab1 in HBE cells transfected with 50nM scrambled siRNA. Error bars represent the standard error of the mean (n ¼ 3).

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relative to scrambled control. When cells were transfected with two hits of Rab1b siRNA at this same concentration, the knockdown of Rab1 was 59.6  8.5. These data reveal that the optimal conditions for efficient silencing of Rab1b in HBE cells include two sequential transfections with complexes composed of 50 nM final concentration of siRNA mixed with 6 ml of HiPerFect. The optimal volume of HiPerFect to be used for each well was previously determined for other siRNA targets (data not shown).

5. SDS-PAGE Analysis of Rab1 Knockdown Lysis Buffer: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 2 mg/ml of EDTA-free protease-inhibitor cocktail (Roche cat. no. 13129100) SDS Sample Buffer: 120 mM, Tris-HCl, pH 7.0, 30% glycerol, 6% SDS, 0.6% bromophenol blue, and 100 mM DTT Following the knockdown procedure described above, cells were transferred to ice and incubated in lysis buffer on ice for 30 min with occasional rocking. The lysates were collected and centrifuged at 14,000
g for 20 min at 4 and the supernatants were collected. The protein concentration in each sample was determined by Bradford assay (Pierce product no. 1856209) using BSA (Pierce product no. 23209) as a standard. Equal amounts of protein (30 mg) for each sample were supplemented with 6
SDS Sample Buffer and boiled for 15 min prior to loading. The proteins are separated on a 12% SDS-PAGE run and subjected to Western blotting with polyclonal anti-Rab1 antibody p68 (Plutner et al., 1991). The detection was done by ECL and the band intensities compared by quantitative analysis using an Alpha Innotech Fluor SP apparatus and accompanying software (Figs. 1.2 and 1.3; see also Fig. 1.1).

6. SDS-PAGE Analysis of CFTR Analysis of CFTR was performed by SDS-PAGE as described for Rab1; however, samples were incubated at 37 for 15 min in SDS sample buffer instead of boiling since CFTR irreversibly aggregates at high temperatures and will not be detected by SDS-PAGE. These samples were separated on 8% SDS-PAGE in order to resolve the ER and Golgi glycoforms from one another. CFTR was detected using the M3A7 ascites raised against the NBD2 domain of CFTR, and band intensities were analyzed as for Rab1. Relative intensities were normalized to percent of maximal intensity and replicates were averaged and compared using a two-tailed t-test (see Figs. 1.2 and 1.3).

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A

Scrambled A

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Rab1b A

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C IB: CFTR B IB: Rab1 B

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Percent maximum (% max)

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Rab1

Figure 1.2 Knockdown of Rab1b affects stability and trafficking of wildtype CFTR. (A) Western blot analysis of CFTR in cells transfected with 50 nM Rab1b or scrambled siRNA. (B) Quantitative analysis of CFTR stability and trafficking in cells transfected with 50nM Rab1b (open bar) or scrambled (solid bar) siRNA.The levels of bands B and C are expressed as a percent of the most intense signal. (n ¼ 3, *p ¼ 0.05) Inset: Magnification of the band B signal to illustrate the effect of Rab1b siRNA.

In order to ensure that the knockdown of Rab1 resulted in the predicted ER-to-Golgi trafficking defect, we assessed the trafficking and stability of both wildtype and DF508 CFTR. A 60% knockdown of Rab1b in cells expressing wildtype CFTR caused a 43.1  6.9% decrease in the levels of band B (ER) and a concomitant 19.3  3.1% decrease in band C (postGolgi) (see Fig. 1.2A and B). Upon ER export of CFTR, there is no accumulation within the Golgi compartment nor in endocytic vesicular storage pools; hence the band C glycoform represents surface CFTR, which has been previously shown to exhibit a half life on the order of 24 h or more (Lukacs et al., 1993). This suggests that any effects on ER-to-Golgi trafficking resulting from Rab1 knockdown would have minimal effect on the surface pool of wildtype CFTR unless these conditions persisted for prolonged periods of time. This observation is confirmed by the fact that we observed a 20% reduction in the band C glycoform while a 50% reduction

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ΔF508 IB: CFTRC B

Scrambled A

B

C

Rab1b A B

C

Percent maximum (% max)

IB: Rab1 Scr.

100

Rab1b

80 60 40 20 0

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Band C

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Figure 1.3 Knockdown of Rab1b affects stability of DF508 CFTR. Quantitative analysis of DF508 CFTR in cells transfected with Rab1b (open bar) or scrambled (closed bar) siRNA.The levels of bands B and C are expressed as a percent of the most intense signal (n ¼ 3, *p ¼ 0.05).

in the band B glycoform is seen. The decreased levels of the ER glycoform of wildtype CFTR is associated with both a decrease in Rab1b synthesis reflecting its loss of message and rapid proteosomal degradation of CFTR, a pathway that prevents accumulation of wildtype CFTR that fails to exit the ER. In order to more completely ascertain the effect of Rab1b depletion on cell surface stability, a pulse chase experiment would be required to follow both trafficking kinetics (conversion from B to C) and the kinetics of degradation of the band C glycoform. The data above confirm that the knockdown of Rab1b using siRNA is able to result in a defect in ER-to-Golgi trafficking of wildtype CFTR in human cell lines. This is supported by the effect of Rab1b knockdown on the stability of DF508 CFTR, which is unable to traffic to the Golgi compartment and mature to the band C glycoform. Under conditions of maximal Rab1b knockdown, there is a 45.2  5.0% decrease in the band B form of DF508 CFTR (see Fig. 1.3).

7. Discussion We have shown that transfection complexes composed of 50 nM siRNA and 6 ml of HiPerFect transfection reagent presented to cells in two separate hits over a 4-day culture period provide the optimal silencing of Rab1 in human bronchial epithelial cells. Although the concentration of siRNA and the amount of transfection reagent, as well as the length of the post-transfection incubation should be optimized for a given siRNA and

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cell line as per manufacturer suggestions, similar protocols can be used to successfully silence a number of other proteins in these same cell lines as well as in human embryonic kidney cells (HEK293) (data not shown). Although some variation in efficiency of silencing is observed for knockdown of different targets, with the correct siRNA probes, it is not unusual to obtain more than 50% of control, often a value sufficient to elicit a meaningful biological effect. More efficient knockdowns (>80%) are likely to yield more pronounced biological effects, and can be observed pending design and choice of siRNA probe. Although monitoring steady-state levels of a protein is sufficient to indicate impact on cell physiology, a protein with a long half-life could have altered functionality in response to multiple epigenetic modifications (phosphorylation, acetylation, etc.), or in the case of membrane-bound proteins, redistribution to different compartments yielding a different functionality(s). Thus, a second measure of efficacy of knockdown is to follow the protein of interest using a pulse-chase protocol. For example, monitoring the kinetic of synthesis of Rab1b would yield a direct measure of translating message. Moreover, in addition to monitoring the steady state levels of both the B and C glycoforms of CFTR, it would have also been useful to follow maturation of de novo synthesized protein via a pulse chase experiment to obtain a more direct read-out of the ER, Golgi, and cell surface trafficking events. The least reliable method in terms of defining knockdown of function is to follow the message level by quantitative RT-PCR given the unknown half-life of a particular target. However, it has the advantage of being rapid, and does not require additional targetspecific reagents. Quantitative RT-PCR can, at minimum, indicate efficacy of the siRNA probe being used, and is useful to monitor the presence of various closely related isoforms (e.g., Rab1a) that could confound interpretation of results if they display overlap in function. In general, it should be kept in mind that alteration of a particular protein activity such as Rab1b by siRNA silencing may have multiple indirect effects on linked pathways over the typical time course of an siRNA experiment. Consequently, results should be interpreted with caution and validated by alternative approaches, and through rescue of silencing by supplementation with a silencing-resistant cDNA construct with altered code encoding the target protein where possible.

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Anderson, M. P., Rich, D. P., Gregory, R. J., Smith, A. E., and Welsh, M. J. (1991b). Generation of cAMP-activated chloride currents by expression of CFTR. Science 251, 679–682. Cao, X., Ballew, N., and Barlowe, C. (1998). Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins. EMBO J. 17, 2156–2165. Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O’Riordan, C. R., and Smith, A. E. (1990). Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827–834. Gurkan, C., Lapp, H., Alory, C., Su, A. I., Hogenesch, J. B., and Balch, W. E. (2005). Large-scale profiling of Rab GTPase trafficking networks: The membrome. Mol. Biol. Cell 16, 3847–3864. Kerem, B., Rommens, J. M., Buchanan, J. A., Markiewicz, D., Cox, T. K., Chakravarti, A., Buchwald, M., and Tsui, L. C. (1989). Identification of the cystic fibrosis gene: Genetic analysis. Science 245, 1073–1080. Lukacs, G. L., Chang, X. B., Bear, C., Kartner, N., Mohamed, A., Riordan, J. R., and Grinstein, S. (1993). The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells. J. Biol. Chem. 268, 21592–21598. Moyer, B. D., Allan, B. B., and Balch, W. E. (2001). Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis—Golgi tethering. Traffic 2, 268–276. Pereira-Leal, J. B., and Seabra, M. C. (2001). Evolution of the Rab family of small GTPbinding proteins. J. Mol. Biol. 313, 889–901. Pfeffer, S., and Aivazian, D. (2004). Targeting Rab GTPases to distinct membrane compartments. Mol. Cell Biol.Nat. Rev. Mol. Cell. Biol. 5, 886–896. Plutner, H., Cox, A. D., Pind, S., Khosravi-Far, R., Bourne, J. R., Schwaninger, R., Der, C. J., and Balch, W. E. (1991). Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments. J. Cell Biol. 115, 31–43. Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., and Chou, J. L. (1989). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066–1073. Rommens, J. M., Iannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., and Hidaka, N. (1989). Identification of the cystic fibrosis gene: Chromosome walking and jumping. Science 245, 1059–1065.