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Protocol
RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans Nicholas J. Palmisano1,2 and Alicia Meléndez1,2,3 1
Department of Biology, Queens College-CUNY, Flushing, New York 11367; 2The Graduate Center, The City University of New York, New York 10016
RNA interference (RNAi) is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of double-stranded RNA (dsRNA). In the nematode Caenorhabditis elegans, RNA inactivation can be used at any specific developmental stage or during adulthood to inhibit a given target gene. Investigators can take advantage of the fact that, in C. elegans, RNAi is unusual in that it is systemic, meaning that dsRNA can spread throughout the animal and can affect virtually all tissues except neurons. Here, we describe a protocol for the most common method to achieve RNAi in C. elegans, which is to feed them bacteria that express dsRNA complementary to a specific target gene. This method has various advantages, including the availability of libraries that essentially cover the whole genome, the ability to treat animals at any developmental stage, and that it is relatively cost effective. We also discuss how RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes, while also highlighting the caveats that must be applied.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agarose (2%–3%) Ampicillin Caenorhabditis elegans strains of interest (larval stage L4 animals) The strains to be analyzed should be well fed on nematode growth media (NGM) agar plates seeded with Escherichia coli bacteria and cultured for at least two generations before the experiment.
Escherichia coli (HT115 expressing double-stranded RNA against target gene under the isopropylthioβ-galactoside [IPTG]-inducible T7 RNA polymerase promoter) LB (Luria-Bertani) liquid medium M9 minimal medium buffer NGM RNA interference (RNAi) agar plates Sodium azide (NaN3; 25 mM) 3
Correspondence: [email protected]
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Equipment
Fluorescence microscope (Zeiss AxioImager A1) Microscope coverslips (1.22 × 13 mm) Microscope slides (75 × 25 × 0.96 mm) Platinum wire pick (standard) Shaker METHOD For a summary of the whole procedure, see Figure 1.
1. Inoculate each bacterial RNAi clone of interest in 5 mL of liquid medium (LB containing 100 mg/ mL ampicillin) overnight at 37˚C in a shaker at 250 rpm. Be aware of the danger of contamination or incorrect clones. A bacterial clone that is isolated from an RNAi library for the first time should be verified to ensure the presence of the correct insert in the plasmid. Glycerol stocks can then be made from the verified colony.
2. Seed experimental RNAi plates with 500 µL of the bacterial liquid culture, and allow them to dry overnight. Allowing plates to dry overnight before adding worms ensures sufficient induction of T7 RNA polymerase activity.
3. Place 30 L4 larval stage animals on RNAi plates and allow animals to ingest bacteria overnight at the desired temperature. Some C. elegans strains are temperature sensitive, and therefore care should be taken when deciding on the temperature at which the experiment should be performed.
4. On the following day, transfer the animals (now adults) to new RNAi plates using 50 to 100 μL of M9 minimal medium buffer and allow them to lay eggs overnight at the desired temperature. Discard the first set of plates (from Step 3).
Inoculate bacterial culture expressing dsRNA and grow overnight at 37° C
Place L4 hermaphrodites on plates and allow them to lay eggs overnight Inoculate bacterial lawn on RNAi plates and let dry overnight at room temperature
Transfer animals to freshly seeded RNAi plates and allow them to lay eggs overnight Analyze animals once the appropriate stage is reached
Collect synchronized L1 progeny with a hatch-off and allow them to reach the developmental stage of interest
FIGURE 1. Flowchart summarizing a protocol for achieving RNAi-mediated inactivation of autophagy genes in the nematode Caenorhabditis elegans. L1, larval stage 1; L4, larval stage 4; RNAi, RNA interference.
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RNAi of Autophagy Genes in C. elegans The adults can be transferred to fresh RNAi plates for two more days (as a third set or a fourth set), to obtain more F1 animals with knockdown of the specific target gene. If desired, F1 progeny treated with RNAi can be tightly synchronized using the wash-off/hatch-off method with M9 minimal medium buffer (see Protocol: Detection of Autophagy Using GFP::LGG-1 as an Autophagy Marker [Palmisano and Meléndez 2015a]).
5. Incubate the second set of plates at the appropriate temperature until the F1 progeny reach the developmental stage of interest. Only analyzing progeny from the second set of plates ensures that the F1 progeny to be analyzed are derived from mothers that ingested the double-stranded RNA (dsRNA). See Troubleshooting.
6. Apply a single drop of molten agarose (2%–3%) onto a microscope slide. Immediately, place another microscope slide on top of the agarose drop to form a thin agarose pad. After the agarose has solidified, remove the top slide. 7. Pipette 3 µL of 25 mM sodium azide onto the center of the agarose pad. Transfer 10–20 animals into the sodium azide drop and immediately apply a coverslip to prevent evaporation of the sodium azide solution. Wait 30 sec until the animals are completely anesthetized, and then perform imaging (see Protocol: Detection of Autophagy Using GFP::LGG-1 as an Autophagy Marker [Palmisano and Meléndez 2015a] and Protocol: Detection of Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation [Palmisano and Meléndez 2015b]). For sample results, see Figure 2. See Troubleshooting.
TROUBLESHOOTING Problem (Step 5): Exposure to a specific RNAi clone results in embryonic lethality. Solution: It might be that the particular gene targeted by RNAi is required during embryogenesis. To
bypass the requirement for the essential gene, treat L1 animals with the specific RNAi clone and analyze animals once the appropriate stage has been reached. Problem (Step 7): Animals submerged in sodium azide do not become anesthetized. Solution: The concentration of sodium azide might be too low. Remake the solution or increase the
concentration, as needed. Problem (Step 7): Knockdown of a particular gene does not give a phenotype. Solution: Consider the following:
•
The specific RNAi bacterial clone might be contaminated with another clone. It is important to ensure that the plasmid contains the correct DNA sequence corresponding to the gene of interest. If contamination is suspected, streak a fresh LB plate (containing 100 mg/mL ampicillin and 10 mg/mL tetracycline) to isolate new bacterial colonies and inoculate a new colony into 5 mL of LB medium, and then proceed with Step 1.
•
The animals might not have been exposed to the RNAi clone for an adequate amount of time. Allow the worms to ingest the bacteria expressing the dsRNA for a longer period of time.
•
To strengthen the effect of the RNAi, seed the plates with the bacteria expressing the dsRNA, allow the bacterial seed to dry, and then add 0.5–1 mM IPTG directly to the bacteria to induce expression. For this change in the experimental method, the NGM RNAi agar plates should be made only with carbenicillin and should not contain IPTG.
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FIGURE 2. Expression of the fluorescent marker GFP::LGG-1 in hypodermal seam cells of daf-2(e1370) mutants of the nematode Caenorhabditis elegans. (A) daf-2(e1370) mutants grown on OP50 Escherichia coli, at 15˚C, display a diffuse localization of GFP::LGG-1. (B) daf-2(e1370) mutants grown on OP50 E. coli, at 25˚C, display an increase in GFP::LGG-1-positive puncta (up to 12 GFP::LGG-1-positive puncta per seam cell) that represent early autophagic structures or autophagosomes. (C ) daf-2(e1370) mutants grown on control RNAi E. coli (transformed with empty vector, L4440), at 25˚C, display the characteristic GFP::LGG-1-positive punctate structures. (D) daf-2(e1370) mutants fed bec-1 RNAi, and raised at 25˚C, display an increase in GFP::LGG-1 expression and large GFP::LGG-1positive aggregates.
DISCUSSION RNAi Enables the Study of Autophagy in Worms
RNAi is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of dsRNA (Fire et al. 1998). In C. elegans, RNA inactivation can be applied at any specific developmental stage, or only during adulthood to avoid developmental requirements for a given target gene. Caenorhabditis elegans is unusual in that RNAi is systemic, meaning that the dsRNA can spread throughout the animal and affect virtually all tissues except neurons (Grishok et al. 2000; Winston et al. 2002). There are multiple ways to deliver dsRNA; however, the most common method used in C. elegans is to feed the animals bacteria that express dsRNA complementary to a specific target gene (Timmons and Fire 1998; Kamath et al. 2001). This method is advantageous because libraries are available that represent most genes in C. elegans (Kamath et al. 2003; Rual et al. 2004), and we have the ability to knock down a given target gene beginning at any developmental stage. The dsRNA can also be administered through injection or soaking; however, these methods require the in vitro preparation of dsRNA and are more time consuming (Ahringer 2006). One problem with the soaking method is that it can also introduce a level of stress as C. elegans do not feed well in liquid media and can undergo starvation (Klass 1977). However, the injection method has been shown to be more efficient in the inactivation of certain target genes (Ahringer 2006). 188
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RNAi of Autophagy Genes in C. elegans
Autophagy genes were shown to be required for the changes associated with dauer morphogenesis in daf-2 dauer constitutive mutants. daf-2 encodes the C. elegans Insulin-like/IGF-1 receptor (IIR). RNAi has been greatly advantageous for the study of autophagy genes in the development of C. elegans. RNAi treatment against unc-51, bec-1, atg-7, lgg-1, and atg-18 (by injection of dsRNA) has been shown to block dauer morphogenesis of daf-2/IIR dauer constitutive mutants, and to result in the appearance of GFP::LGG-1-positive protein aggregates in hypodermal seam cells (Meléndez et al. 2003). Defects in dauer development and GFP::LGG-1 expression are also observed after feeding animals RNAi against bec-1 or against several other autophagy genes. However, we find that the phenotypes observed after feeding the RNAi bacteria may not be as penetrant as those after the dsRNA is delivered by injection. Although the exact nature of the GFP::LGG-1 aggregates is not clear, they have been suggested to be polyubiquitylated proteins that accumulate as a result of ineffective lysosomal degradation (Szeto et al. 2006). Therefore, GFP::LGG-1 localization should be carefully interpreted when using RNAi. Knockdown of bec-1 gene activity (by injection or feeding), and knockdown of atg-12 and atg-7 gene activity (by feeding only), has shown that these autophagy genes are also required for the longevity phenotype of daf-2/IIR mutants (Meléndez et al. 2003; Hars et al. 2007; Hansen et al. 2008). RNAi, by feeding during adulthood, against vps-34, atg-7, unc-51, atg-18, lgg-1, or bec-1 has also been shown to decrease the life span extension of dietary-restricted eat-2 mutants (Hansen et al. 2008) and, more recently, of germline-less glp-1/Notch mutants (Lapierre et al. 2011). The decrease in life span is not due to the effects of RNAi treatment during development, which can lead to decreased longevity as RNAi treatment was conducted during adulthood (Hansen et al. 2008; Meléndez et al. 2008). In conclusion, RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes. For a description of the methods applicable to longevity assays, the reader is referred elsewhere (Meléndez et al. 2008). Data Analysis
Confirmation that gene knockdown has occurred can be easily determined by measuring a reduction in mRNA levels of the target gene by reverse transcription polymerase chain reaction (PCR), or by measuring a reduction in protein levels by western blot analysis (Ahringer 2006). In addition, the success of an RNAi experiment can be confirmed through the use of appropriate positive and negative controls. For example, when using strains that contain a green-fluorescent protein (GFP)-tagged fluorescent reporter, a positive control that can be used is to feed animals with RNAi-expressing bacteria targeting GFP (Timmons et al. 2001). After treatment with GFP RNAi, the expression of any GFP reporter should decrease significantly. A more formal negative control for RNAi is to feed animals with bacteria transformed with the empty vector control L4440 (Timmons and Fire 1998). Results obtained after RNAi treatment against a gene of interest should be compared with the results obtained after RNAi against the negative control with empty vector to determine whether the RNAi clone tested is the direct cause for any observed phenotype. The effects of RNAi can be further enhanced by using certain mutations that have been shown to increase the sensitivity of animals to RNAi, such as mutations in the lin-35, lin-15b, eri-1, and rrf-3 loci (Simmer et al. 2002; Kennedy et al. 2004; Wang et al. 2005; Lehner et al. 2006; Schmitz et al. 2007). To investigate the specific tissue that requires the activity of a the target gene, by RNAi, several strains can be used that carry mutations that confer RNAi resistance in particular tissues (Smardon et al. 2000; Sijen et al. 2001; Kumsta and Hansen 2012). Furthermore, as neurons are commonly known to be refractory to RNAi treatment, to treat neurons by RNAi requires a strain with neuronal expression of SID-1, a transmembrane protein required for systemic RNAi (Calixto et al. 2010). When expressed in neurons, SID-1 increases the response of those neurons to RNAi; however, neuronal expression of sid1 has been shown to decrease the effects of nonneuronal RNAi (Calixto et al. 2010), which has to be taken into consideration when evaluating gene knockdown in both neuronal and nonneuronal tissues. Additionally, expression of sid-1(+) from a cell-specific promoter in a sid-1(–) mutant can be used as a Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086520
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cell-specific method of feeding RNAi and limiting the effect of gene knockdown to specific cell types (i.e., neurons) (Calixto et al. 2010). In C. elegans, ego-1, and rrf-1 encode RNA-dependent RNA polymerases, with tissue-specific RNAi-processing function—ego-1 partially targets the germline and rrf-1 targets the somatic tissues (Smardon et al. 2000; Sijen et al. 2001; Suzuki et al. 2004; Qadota et al. 2007; Kumsta and Hansen 2012). Thus, to enquire whether a particular gene acts in somatic tissues, or in the germline, one can use a strain containing a mutation in the rrf-1 locus (Sijen et al. 2001). Two mutations of rrf-1, pk1417, and ok589, are available and have been shown to have no effect on life span or thermotolerance (Kumsta and Hansen 2012). However, care should be taken when using rrf-1 mutants as these mutants have been found capable of processing RNAi in somatic tissues, particularly in the intestine and a subset of hypodermal cells (Kumsta and Hansen 2012). Another consideration is whether rrf-1 exerts any phenotype in the process being studied as it has been shown that the rrf-1 mutation induces the expression of several transgenes and increases the expression of sod-2, a DAF-16/FOXO transcription target (Kumsta and Hansen 2012). Another method that has been previously used to achieve RNAi in specific tissues is to use an rde-1 mutant strain that is resistant to RNAi (Tabara et al. 1999) and to express the wild-type rde-1 cDNA under the control of tissue-specific promoters to achieve RNAi sensitivity in the tissues that express rde-1 (Suzuki et al. 2004; Qadota et al. 2007). However, several considerations have to be made when using the tissue-specific promoters as they might not rescue completely and they can sometimes be expressed in other tissues. For genes that express and/or function in multiple tissues, the assay of tissue-specific RNAi can be very helpful in distinguishing the activity of that gene in a particular tissue or cell type. There are multiple phenotypes that have been shown to depend on autophagy gene activity and thus can be used to determine whether a particular gene of interest functions in autophagy. One can treat daf-2/IIR mutants that express the GFP::LGG-1 reporter shown to label structures corresponding to autophagosomes, with RNAi against the gene of interest and evaluate whether the RNAi treatment affects dauer formation or the induction of GFP::LGG-1-positive punctate structures in seam cells. When conducting such an experiment, RNAi specific to autophagy genes, such as bec-1, unc-51, or atg-18, should be used as positive controls. RNAi against bec-1, unc-51, or atg-18 has been shown to increase the diffuse expression of GFP::LGG-1 and also results in the formation of GFP::LGG-1 protein aggregates (see Fig. 2) (Meléndez et al. 2003). However, the nature of the GFP::LGG-1 aggregates that arise because of inhibition, or knockdown of autophagy gene activity, is not fully understood. Thus, the formation of GFP::LGG-1 protein aggregates that arise as a result of knocking down the gene of interest should be further examined to determine where the gene of interest acts in the autophagy pathway. A next step would be to investigate whether the specific RNAi treatment affects the formation of autophagosomes, lysosomal degradation, or the accumulation of polyubiquitylated proteins in such a way as to exceed the degradative capacity of the lysosome.
Concluding Remarks
RNAi is sometimes more advantageous to study gene function than mutations, as pleiotropic effects during development can be avoided by treating animals as adults or at a specific developmental stage. For example, in the case of bec-1, deletion mutants that lack both maternal and zygotic bec-1 activity are embryonic lethal; however, bec-1 RNAi animals live until young adulthood and display severe vacuolization and incoordination (Ruck et al. 2011). Therefore, RNAi provides a means to study gene function by circumventing some of the drawbacks associated with strong loss-of-function mutations. When investigating whether a particular gene functions in autophagy, daf-2 mutants expressing GFP::LGG-1 can be treated with RNAi specific for the gene of interest and evaluated for changes in the expression pattern of GFP::LGG-1. Other mutant backgrounds, such as eat-2 dietary-restricted or glp1 germline-less mutants, can also be used for investigating whether a particular gene functions in 190
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RNAi of Autophagy Genes in C. elegans
autophagy; however, as for daf-2 mutants, appropriate controls are required to make accurate conclusions and comparisons from the results obtained.
RECIPES LB (Luria-Bertani) Liquid Medium
Reagent
Amount to add
H2O Tryptone NaCl Yeast extract
950 mL 10 g 10 g 5g
Combine the reagents and shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (0.2 mL). Adjust the final volume of the solution to 1 L with H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. M9 Minimal Medium Buffer
Reagent
Concentration (mM)
KH2PO4 Na2HPO4 NaCl MgSO4
22 mM 22 mM 85 mM 1 mM
Autoclave for 15 min on liquid cycle. Allow medium to cool and store at room temperature. NGM RNAi Agar Plates
68 g Bacto agar powder 12 g NaCl 10 g Bacto peptone 4 mL cholesterol (5 mg/mL in ethanol) 3.9 L deionized water 4 mL CaCl2 (1 M) 4 mL MgSO4 (1 M) 100 mL KPO4 (1 M) 50 mg/mL carbenicilin 1 M Isopropyl B-D-1-thiogalactopyranoside (IPTG) To prepare 4 L of normal growth media (NGM), mix the first five ingredients, and autoclave for 70 min on liquid cycle. Ensure that the agar is dissolved, let cool, and then add the last five ingredients. Pour the plates (these can be stored for up to 6 wk at 4˚C).
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Grishok A, Tabara H, Mello CC. 2000. Genetic requirements for inheritance of RNAi in C. elegans. Science 287: 2494–2497. Hansen M, Chandra A, Mitic LL, Onken B, Driscoll M, Kenyon C. 2008. A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. PLoS Genet 4: e24. Hars ES, Qi H, Ryazanov AG, Jin S, Cai L, Hu C, Liu LF. 2007. Autophagy regulates ageing in C. elegans. Autophagy 3: 93–95. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J. 2001. Effectiveness of specific RNA-mediated interference through in-
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gested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2: RESEARCH0002. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, et al. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237. Kennedy S, Wang D, Ruvkun G. 2004. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427: 645–649. Klass MR. 1977. Aging in the nematode Caenorhabditis elegans: Major biological and environmental factors influencing life span. Mech Ageing Dev 6: 413–429. Kumsta C, Hansen M. 2012. C. elegans rrf-1 mutations maintain RNAi efficiency in the soma in addition to the germline. PLoS ONE 7: e35428. Lapierre LR, Gelino S, Meléndez A, Hansen M. 2011. Autophagy and lipid metabolism coordinately modulate life span in germline-less C. elegans. Curr Biol 21: 1507–1514. Lehner B, Calixto A, Crombie C, Tischler J, Fortunato A, Chalfie M, Fraser AG. 2006. Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference. Genome Biol 7: R4. Meléndez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B. 2003. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301: 1387–1391. Meléndez A, Hall DH, Hansen M. 2008. Monitoring the role of autophagy in C. elegans aging. Methods Enzymol 451: 493–520. Palmisano NJ, Meléndez A. 2015a. Detection of autophagy using GFP::LGG1 as an autophagy marker. Cold Spring Harb Protoc doi: 10.1101/pdb .prot086496. Palmisano NJ, Meléndez A. 2015b. Visualization of autophagy in Caenorhabditis elegans embryos. Cold Spring Harb Protoc doi: 10.1101/pdb .prot086504. Qadota H, Inoue M, Hikita T, Koppen M, Hardin JD, Amano M, Moerman DG, Kaibuchi K. 2007. Establishment of a tissue-specific RNAi system in C. elegans. Gene 400: 166–173. Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane-Kishikawa T, Vandenhaute J, Orkin SH, Hill DE, van den Heuvel S, et al. 2004. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14: 2162–2168. Ruck A, Attonito J, Garces KT, Nunez L, Palmisano NJ, Rubel Z, Bai Z, Nguyen KC, Sun L, Grant BD, et al. 2011. The Atg6/Vps30/Beclin1
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Protocol
RNAi-Mediated Inactivation of Autophagy Genes in Caenorhabditis elegans Nicholas J. Palmisano1,2 and Alicia Meléndez1,2,3 1
Department of Biology, Queens College-CUNY, Flushing, New York 11367; 2The Graduate Center, The City University of New York, New York 10016
RNA interference (RNAi) is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of double-stranded RNA (dsRNA). In the nematode Caenorhabditis elegans, RNA inactivation can be used at any specific developmental stage or during adulthood to inhibit a given target gene. Investigators can take advantage of the fact that, in C. elegans, RNAi is unusual in that it is systemic, meaning that dsRNA can spread throughout the animal and can affect virtually all tissues except neurons. Here, we describe a protocol for the most common method to achieve RNAi in C. elegans, which is to feed them bacteria that express dsRNA complementary to a specific target gene. This method has various advantages, including the availability of libraries that essentially cover the whole genome, the ability to treat animals at any developmental stage, and that it is relatively cost effective. We also discuss how RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes, while also highlighting the caveats that must be applied.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Agarose (2%–3%) Ampicillin Caenorhabditis elegans strains of interest (larval stage L4 animals) The strains to be analyzed should be well fed on nematode growth media (NGM) agar plates seeded with Escherichia coli bacteria and cultured for at least two generations before the experiment.
Escherichia coli (HT115 expressing double-stranded RNA against target gene under the isopropylthioβ-galactoside [IPTG]-inducible T7 RNA polymerase promoter) LB (Luria-Bertani) liquid medium M9 minimal medium buffer NGM RNA interference (RNAi) agar plates Sodium azide (NaN3; 25 mM) 3
Correspondence: [email protected]
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Equipment
Fluorescence microscope (Zeiss AxioImager A1) Microscope coverslips (1.22 × 13 mm) Microscope slides (75 × 25 × 0.96 mm) Platinum wire pick (standard) Shaker METHOD For a summary of the whole procedure, see Figure 1.
1. Inoculate each bacterial RNAi clone of interest in 5 mL of liquid medium (LB containing 100 mg/ mL ampicillin) overnight at 37˚C in a shaker at 250 rpm. Be aware of the danger of contamination or incorrect clones. A bacterial clone that is isolated from an RNAi library for the first time should be verified to ensure the presence of the correct insert in the plasmid. Glycerol stocks can then be made from the verified colony.
2. Seed experimental RNAi plates with 500 µL of the bacterial liquid culture, and allow them to dry overnight. Allowing plates to dry overnight before adding worms ensures sufficient induction of T7 RNA polymerase activity.
3. Place 30 L4 larval stage animals on RNAi plates and allow animals to ingest bacteria overnight at the desired temperature. Some C. elegans strains are temperature sensitive, and therefore care should be taken when deciding on the temperature at which the experiment should be performed.
4. On the following day, transfer the animals (now adults) to new RNAi plates using 50 to 100 μL of M9 minimal medium buffer and allow them to lay eggs overnight at the desired temperature. Discard the first set of plates (from Step 3).
Inoculate bacterial culture expressing dsRNA and grow overnight at 37° C
Place L4 hermaphrodites on plates and allow them to lay eggs overnight Inoculate bacterial lawn on RNAi plates and let dry overnight at room temperature
Transfer animals to freshly seeded RNAi plates and allow them to lay eggs overnight Analyze animals once the appropriate stage is reached
Collect synchronized L1 progeny with a hatch-off and allow them to reach the developmental stage of interest
FIGURE 1. Flowchart summarizing a protocol for achieving RNAi-mediated inactivation of autophagy genes in the nematode Caenorhabditis elegans. L1, larval stage 1; L4, larval stage 4; RNAi, RNA interference.
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RNAi of Autophagy Genes in C. elegans The adults can be transferred to fresh RNAi plates for two more days (as a third set or a fourth set), to obtain more F1 animals with knockdown of the specific target gene. If desired, F1 progeny treated with RNAi can be tightly synchronized using the wash-off/hatch-off method with M9 minimal medium buffer (see Protocol: Detection of Autophagy Using GFP::LGG-1 as an Autophagy Marker [Palmisano and Meléndez 2015a]).
5. Incubate the second set of plates at the appropriate temperature until the F1 progeny reach the developmental stage of interest. Only analyzing progeny from the second set of plates ensures that the F1 progeny to be analyzed are derived from mothers that ingested the double-stranded RNA (dsRNA). See Troubleshooting.
6. Apply a single drop of molten agarose (2%–3%) onto a microscope slide. Immediately, place another microscope slide on top of the agarose drop to form a thin agarose pad. After the agarose has solidified, remove the top slide. 7. Pipette 3 µL of 25 mM sodium azide onto the center of the agarose pad. Transfer 10–20 animals into the sodium azide drop and immediately apply a coverslip to prevent evaporation of the sodium azide solution. Wait 30 sec until the animals are completely anesthetized, and then perform imaging (see Protocol: Detection of Autophagy Using GFP::LGG-1 as an Autophagy Marker [Palmisano and Meléndez 2015a] and Protocol: Detection of Autophagy in Caenorhabditis elegans Embryos Using Markers of P Granule Degradation [Palmisano and Meléndez 2015b]). For sample results, see Figure 2. See Troubleshooting.
TROUBLESHOOTING Problem (Step 5): Exposure to a specific RNAi clone results in embryonic lethality. Solution: It might be that the particular gene targeted by RNAi is required during embryogenesis. To
bypass the requirement for the essential gene, treat L1 animals with the specific RNAi clone and analyze animals once the appropriate stage has been reached. Problem (Step 7): Animals submerged in sodium azide do not become anesthetized. Solution: The concentration of sodium azide might be too low. Remake the solution or increase the
concentration, as needed. Problem (Step 7): Knockdown of a particular gene does not give a phenotype. Solution: Consider the following:
•
The specific RNAi bacterial clone might be contaminated with another clone. It is important to ensure that the plasmid contains the correct DNA sequence corresponding to the gene of interest. If contamination is suspected, streak a fresh LB plate (containing 100 mg/mL ampicillin and 10 mg/mL tetracycline) to isolate new bacterial colonies and inoculate a new colony into 5 mL of LB medium, and then proceed with Step 1.
•
The animals might not have been exposed to the RNAi clone for an adequate amount of time. Allow the worms to ingest the bacteria expressing the dsRNA for a longer period of time.
•
To strengthen the effect of the RNAi, seed the plates with the bacteria expressing the dsRNA, allow the bacterial seed to dry, and then add 0.5–1 mM IPTG directly to the bacteria to induce expression. For this change in the experimental method, the NGM RNAi agar plates should be made only with carbenicillin and should not contain IPTG.
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FIGURE 2. Expression of the fluorescent marker GFP::LGG-1 in hypodermal seam cells of daf-2(e1370) mutants of the nematode Caenorhabditis elegans. (A) daf-2(e1370) mutants grown on OP50 Escherichia coli, at 15˚C, display a diffuse localization of GFP::LGG-1. (B) daf-2(e1370) mutants grown on OP50 E. coli, at 25˚C, display an increase in GFP::LGG-1-positive puncta (up to 12 GFP::LGG-1-positive puncta per seam cell) that represent early autophagic structures or autophagosomes. (C ) daf-2(e1370) mutants grown on control RNAi E. coli (transformed with empty vector, L4440), at 25˚C, display the characteristic GFP::LGG-1-positive punctate structures. (D) daf-2(e1370) mutants fed bec-1 RNAi, and raised at 25˚C, display an increase in GFP::LGG-1 expression and large GFP::LGG-1positive aggregates.
DISCUSSION RNAi Enables the Study of Autophagy in Worms
RNAi is a process that results in the sequence-specific silencing of endogenous mRNA through the introduction of dsRNA (Fire et al. 1998). In C. elegans, RNA inactivation can be applied at any specific developmental stage, or only during adulthood to avoid developmental requirements for a given target gene. Caenorhabditis elegans is unusual in that RNAi is systemic, meaning that the dsRNA can spread throughout the animal and affect virtually all tissues except neurons (Grishok et al. 2000; Winston et al. 2002). There are multiple ways to deliver dsRNA; however, the most common method used in C. elegans is to feed the animals bacteria that express dsRNA complementary to a specific target gene (Timmons and Fire 1998; Kamath et al. 2001). This method is advantageous because libraries are available that represent most genes in C. elegans (Kamath et al. 2003; Rual et al. 2004), and we have the ability to knock down a given target gene beginning at any developmental stage. The dsRNA can also be administered through injection or soaking; however, these methods require the in vitro preparation of dsRNA and are more time consuming (Ahringer 2006). One problem with the soaking method is that it can also introduce a level of stress as C. elegans do not feed well in liquid media and can undergo starvation (Klass 1977). However, the injection method has been shown to be more efficient in the inactivation of certain target genes (Ahringer 2006). 188
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RNAi of Autophagy Genes in C. elegans
Autophagy genes were shown to be required for the changes associated with dauer morphogenesis in daf-2 dauer constitutive mutants. daf-2 encodes the C. elegans Insulin-like/IGF-1 receptor (IIR). RNAi has been greatly advantageous for the study of autophagy genes in the development of C. elegans. RNAi treatment against unc-51, bec-1, atg-7, lgg-1, and atg-18 (by injection of dsRNA) has been shown to block dauer morphogenesis of daf-2/IIR dauer constitutive mutants, and to result in the appearance of GFP::LGG-1-positive protein aggregates in hypodermal seam cells (Meléndez et al. 2003). Defects in dauer development and GFP::LGG-1 expression are also observed after feeding animals RNAi against bec-1 or against several other autophagy genes. However, we find that the phenotypes observed after feeding the RNAi bacteria may not be as penetrant as those after the dsRNA is delivered by injection. Although the exact nature of the GFP::LGG-1 aggregates is not clear, they have been suggested to be polyubiquitylated proteins that accumulate as a result of ineffective lysosomal degradation (Szeto et al. 2006). Therefore, GFP::LGG-1 localization should be carefully interpreted when using RNAi. Knockdown of bec-1 gene activity (by injection or feeding), and knockdown of atg-12 and atg-7 gene activity (by feeding only), has shown that these autophagy genes are also required for the longevity phenotype of daf-2/IIR mutants (Meléndez et al. 2003; Hars et al. 2007; Hansen et al. 2008). RNAi, by feeding during adulthood, against vps-34, atg-7, unc-51, atg-18, lgg-1, or bec-1 has also been shown to decrease the life span extension of dietary-restricted eat-2 mutants (Hansen et al. 2008) and, more recently, of germline-less glp-1/Notch mutants (Lapierre et al. 2011). The decrease in life span is not due to the effects of RNAi treatment during development, which can lead to decreased longevity as RNAi treatment was conducted during adulthood (Hansen et al. 2008; Meléndez et al. 2008). In conclusion, RNAi specific to autophagy genes has proven to be an excellent method to study the role of these genes in autophagy, as well as other cellular and developmental processes. For a description of the methods applicable to longevity assays, the reader is referred elsewhere (Meléndez et al. 2008). Data Analysis
Confirmation that gene knockdown has occurred can be easily determined by measuring a reduction in mRNA levels of the target gene by reverse transcription polymerase chain reaction (PCR), or by measuring a reduction in protein levels by western blot analysis (Ahringer 2006). In addition, the success of an RNAi experiment can be confirmed through the use of appropriate positive and negative controls. For example, when using strains that contain a green-fluorescent protein (GFP)-tagged fluorescent reporter, a positive control that can be used is to feed animals with RNAi-expressing bacteria targeting GFP (Timmons et al. 2001). After treatment with GFP RNAi, the expression of any GFP reporter should decrease significantly. A more formal negative control for RNAi is to feed animals with bacteria transformed with the empty vector control L4440 (Timmons and Fire 1998). Results obtained after RNAi treatment against a gene of interest should be compared with the results obtained after RNAi against the negative control with empty vector to determine whether the RNAi clone tested is the direct cause for any observed phenotype. The effects of RNAi can be further enhanced by using certain mutations that have been shown to increase the sensitivity of animals to RNAi, such as mutations in the lin-35, lin-15b, eri-1, and rrf-3 loci (Simmer et al. 2002; Kennedy et al. 2004; Wang et al. 2005; Lehner et al. 2006; Schmitz et al. 2007). To investigate the specific tissue that requires the activity of a the target gene, by RNAi, several strains can be used that carry mutations that confer RNAi resistance in particular tissues (Smardon et al. 2000; Sijen et al. 2001; Kumsta and Hansen 2012). Furthermore, as neurons are commonly known to be refractory to RNAi treatment, to treat neurons by RNAi requires a strain with neuronal expression of SID-1, a transmembrane protein required for systemic RNAi (Calixto et al. 2010). When expressed in neurons, SID-1 increases the response of those neurons to RNAi; however, neuronal expression of sid1 has been shown to decrease the effects of nonneuronal RNAi (Calixto et al. 2010), which has to be taken into consideration when evaluating gene knockdown in both neuronal and nonneuronal tissues. Additionally, expression of sid-1(+) from a cell-specific promoter in a sid-1(–) mutant can be used as a Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot086520
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cell-specific method of feeding RNAi and limiting the effect of gene knockdown to specific cell types (i.e., neurons) (Calixto et al. 2010). In C. elegans, ego-1, and rrf-1 encode RNA-dependent RNA polymerases, with tissue-specific RNAi-processing function—ego-1 partially targets the germline and rrf-1 targets the somatic tissues (Smardon et al. 2000; Sijen et al. 2001; Suzuki et al. 2004; Qadota et al. 2007; Kumsta and Hansen 2012). Thus, to enquire whether a particular gene acts in somatic tissues, or in the germline, one can use a strain containing a mutation in the rrf-1 locus (Sijen et al. 2001). Two mutations of rrf-1, pk1417, and ok589, are available and have been shown to have no effect on life span or thermotolerance (Kumsta and Hansen 2012). However, care should be taken when using rrf-1 mutants as these mutants have been found capable of processing RNAi in somatic tissues, particularly in the intestine and a subset of hypodermal cells (Kumsta and Hansen 2012). Another consideration is whether rrf-1 exerts any phenotype in the process being studied as it has been shown that the rrf-1 mutation induces the expression of several transgenes and increases the expression of sod-2, a DAF-16/FOXO transcription target (Kumsta and Hansen 2012). Another method that has been previously used to achieve RNAi in specific tissues is to use an rde-1 mutant strain that is resistant to RNAi (Tabara et al. 1999) and to express the wild-type rde-1 cDNA under the control of tissue-specific promoters to achieve RNAi sensitivity in the tissues that express rde-1 (Suzuki et al. 2004; Qadota et al. 2007). However, several considerations have to be made when using the tissue-specific promoters as they might not rescue completely and they can sometimes be expressed in other tissues. For genes that express and/or function in multiple tissues, the assay of tissue-specific RNAi can be very helpful in distinguishing the activity of that gene in a particular tissue or cell type. There are multiple phenotypes that have been shown to depend on autophagy gene activity and thus can be used to determine whether a particular gene of interest functions in autophagy. One can treat daf-2/IIR mutants that express the GFP::LGG-1 reporter shown to label structures corresponding to autophagosomes, with RNAi against the gene of interest and evaluate whether the RNAi treatment affects dauer formation or the induction of GFP::LGG-1-positive punctate structures in seam cells. When conducting such an experiment, RNAi specific to autophagy genes, such as bec-1, unc-51, or atg-18, should be used as positive controls. RNAi against bec-1, unc-51, or atg-18 has been shown to increase the diffuse expression of GFP::LGG-1 and also results in the formation of GFP::LGG-1 protein aggregates (see Fig. 2) (Meléndez et al. 2003). However, the nature of the GFP::LGG-1 aggregates that arise because of inhibition, or knockdown of autophagy gene activity, is not fully understood. Thus, the formation of GFP::LGG-1 protein aggregates that arise as a result of knocking down the gene of interest should be further examined to determine where the gene of interest acts in the autophagy pathway. A next step would be to investigate whether the specific RNAi treatment affects the formation of autophagosomes, lysosomal degradation, or the accumulation of polyubiquitylated proteins in such a way as to exceed the degradative capacity of the lysosome.
Concluding Remarks
RNAi is sometimes more advantageous to study gene function than mutations, as pleiotropic effects during development can be avoided by treating animals as adults or at a specific developmental stage. For example, in the case of bec-1, deletion mutants that lack both maternal and zygotic bec-1 activity are embryonic lethal; however, bec-1 RNAi animals live until young adulthood and display severe vacuolization and incoordination (Ruck et al. 2011). Therefore, RNAi provides a means to study gene function by circumventing some of the drawbacks associated with strong loss-of-function mutations. When investigating whether a particular gene functions in autophagy, daf-2 mutants expressing GFP::LGG-1 can be treated with RNAi specific for the gene of interest and evaluated for changes in the expression pattern of GFP::LGG-1. Other mutant backgrounds, such as eat-2 dietary-restricted or glp1 germline-less mutants, can also be used for investigating whether a particular gene functions in 190
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RNAi of Autophagy Genes in C. elegans
autophagy; however, as for daf-2 mutants, appropriate controls are required to make accurate conclusions and comparisons from the results obtained.
RECIPES LB (Luria-Bertani) Liquid Medium
Reagent
Amount to add
H2O Tryptone NaCl Yeast extract
950 mL 10 g 10 g 5g
Combine the reagents and shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (0.2 mL). Adjust the final volume of the solution to 1 L with H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. M9 Minimal Medium Buffer
Reagent
Concentration (mM)
KH2PO4 Na2HPO4 NaCl MgSO4
22 mM 22 mM 85 mM 1 mM
Autoclave for 15 min on liquid cycle. Allow medium to cool and store at room temperature. NGM RNAi Agar Plates
68 g Bacto agar powder 12 g NaCl 10 g Bacto peptone 4 mL cholesterol (5 mg/mL in ethanol) 3.9 L deionized water 4 mL CaCl2 (1 M) 4 mL MgSO4 (1 M) 100 mL KPO4 (1 M) 50 mg/mL carbenicilin 1 M Isopropyl B-D-1-thiogalactopyranoside (IPTG) To prepare 4 L of normal growth media (NGM), mix the first five ingredients, and autoclave for 70 min on liquid cycle. Ensure that the agar is dissolved, let cool, and then add the last five ingredients. Pour the plates (these can be stored for up to 6 wk at 4˚C).
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