High-Throughput RNAi Screening for N-Glycosylation Dependent Loci in Caenorhabditis elegans

C H A P T E R

T W E N T Y- T W O

High-Throughput RNAi Screening for N-Glycosylation Dependent Loci in Caenorhabditis elegans Weston B. Struwe* and Charles E. Warren† Contents 478 479 480 480 482 482 482 483 486 486 489 492

1. 2. 3. 4.

Overview N-glycosylation in C. elegans RNAi in C. elegans C. elegans Strains, Culturing and RNAi Methods 4.1. Effect of tunicamycin on C. elegans phenotypes 4.2. Tunicamycin-lethality is dose dependent 4.3. Tunicamycin treatment varies among strains 4.4. Establishment of tunicamycin-hypersensitive genes 4.5. Tunicamycin does not alter RNAi effectiveness 5. Genome-wide RNAi Screen 6. Discussion References

Abstract The attachment of oligosaccharides to the amide nitrogen of asparagine side chains on proteins is a fundamental process occurring in all metazoans. This process, known as N-glycosylation, is complex and is achieved by the precise interactions of various cellular components. The initial stage of N-glycan biosynthesis is preserved among eukaryotes, and defective enzymes or components in this pathway cause congenital disorders of glycosylation type I (CDG-I) in humans. This disease is rare but exceedingly life-threatening with no known cure. Paramount to CDG treatment and care is understanding the mechanisms of N-glycosylation and factors that influence the pathology of the disease, both of which are not completely known. Here we outline a novel technique to model a CDG-I-like condition and identify genes that are vital for healthy glycosylation in Caenorhabditis elegans. C. elegans is a well-established model for understanding * National Institute for Bioprocessing Research and Training, Dublin-Oxford Glycobiology Group, Conway Institute for Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin, Ireland Department of Biochemistry and Molecular Biology and Program in Genetics, University of New Hampshire, Durham, NH, USA

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Methods in Enzymology, Volume 480 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)80021-7

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2010 Elsevier Inc. All rights reserved.

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the complexity of glycosylation in development and disease. Although C. elegans N-glycan structures are dissimilar to that observed in higher eukaryotes, they contain over 150 gene homologs that are directly involved in glycosylation. Moreover, the annotated genome of C. elegans, its susceptibility to genetic silencing and its recognizable phenotypes, is a suitable model to dissect the complex phenomenon of glycosylation and identify genes that are required for N-glycan biosynthesis.

1. Overview N-linked glycosylation is an essential protein posttranslational modification that occurs in all eukaryotes. The early stages of N-glycan biosynthesis, namely the formation of the lipid-linked oligosaccharide (LLO) structure in the endoplasmic reticulum, is conserved among Saccharomyces cerevisiae, Caenorhabditis elegans, and vertebrates (Altmann et al., 2001; Huffaker and Robbins, 1982). The LLO consists of a Glc3Man9GlcNAc2 molecule linked to a dolichol pyrophosphate, and defects in enzymes responsible for its synthesis lead to congenital disorders of glycosylation type I (CDG-I) in humans. The biosynthetic pathway for glycoprotein formation is quite well understood and has provided a basis for establishing the etiology of CDG (Schollen et al., 2004). However, there are still cases of CDG with unknown etiology and the mechanistic consequences of defective glycoprotein synthesis are very poorly understood (Prietsch et al., 2002). Loss-of-function defects in the LLO biosynthetic pathway are embryonic lethal, patients with milder genotypes are born but are very ill, presenting with defects in virtually every body system. Because so many structures and functions are compromised, the glycoproteins that cause lethality or particular symptoms cannot be resolved. For this reason, it is vital to understand all factors that contribute to N-glycosylation that may affect the severity of CDG illnesses. Dissecting the complex biosynthesis of glycosylation is not uncomplicated, but here we present a novel approach to identify genes in C. elegans that contribute to the severity of a CDG-I-like disorder. We were able to identify genes in C. elegans that require the formation of the LLO precursor molecule to function properly and generate wild-type phenotypes. These genes products could originate from four hypothetical categories: (1) polypeptides that require glycans for function, (2) polypeptides that require glycans for structure, (3) genes responsible for glycoprotein maturation (i.e., unfolded protein response, secretion, or Golgi function), (4) genes that are involved in the lipid-linked oligosaccharide assembly pathway. To explore this hypothesis, RNAi, in combination with tunicamycin, a glycosylation inhibitor, was used in a genome-wide screen to identify genes that are hypersensitive to a mild dose of tunicamycin.

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2. N-glycosylation in C. elegans The free-living nematode C. elegans is a powerful resource for studying complex developmental topics and provides a robust model system to examine various human diseases. In addition to the ease of maintenance and manipulability C. elegans provides, it is the most understood multicellular eukaryotic animal to date. The complete cell lineage has been mapped and cell fates remain invariant between individuals (Lambie, 2002). C. elegans is a simple organism that comprises only 959 cells, but boasts of feeding, nervous, muscle, and reproductive physiological systems found in higher eukaryotes. More recently, the glycome of C. elegans has been studied and characterized (Cipollo et al., 2002, 2005; Hanneman et al., 2006; Haslam and Dell, 2003; Natsuka et al., 2002). Considering the simple anatomy of C. elegans, its N-glycan composition is extensive with over 100 structures present in the wild-type N2 Bristol strain (Paschinger et al., 2008). The annotated glycome of C. elegans, in addition to its well-characterized physiology, provides a platform to investigate the factors that affect N-glycosylation phenotypes. Much of the progress in our understanding the etiology of CDG has come from genetic model systems, particularly S. cerevisiae and mice (Aebi and Hennet, 2001). C. elegans shares many sequence similarities to mammalian genes involved in the assembly, processing, and modifications of a variety of glycans (Schachter, 2004). Specifically, the early stages of N-glycoprotein biosynthesis are conserved, making C. elegans a suitable model to identify genes that modify CDG-I phenotypes (Lehle et al., 2006). However, the later stages of glycoconjugate synthesis are much more complex in vertebrates, especially mammals. C. elegans do share similarities with vertebrate N- and Oglycans in terms of their core structures, but the majority of the differences are found in molecular size and terminal elaborations. The most notable difference between C. elegans and higher animals is the lack of sialic acid residues in C. elegans glycans (Paschinger et al., 2008). C. elegans have some biosynthetic components required for the synthesis of complex mammalian-type glycans, namely GnT-I (N-acetylglucosamine transferase-I), GnT-II, and GnT-V (Chen et al., 1999, 2002; Warren et al., 2002). Despite the identification of such enzymes, complex and hybrid Nglycans are either absent or present at low levels in C. elegans (Cipollo et al., 2002; Natsuka et al., 2002). However, the occurrence of complex glycan has been found at different developmental stages and may be only present to govern development (Cipollo et al., 2005). Additionally, C. elegans synthesize N-glycans having terminal fucose residues, similar to Lewis antigens found in humans. The C. elegans glycome predominately contains high mannose (Man5-9 GlcNAc2), paucimannose (Man3-4GlcNAc2), high-fucose (attached at the

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antennae or core), truncated complex (Man3GlcNAc3), O-methylated (OMe), and phosphorylcholine (PC)-substituted glycans (Cipollo et al., 2004, 2005; Hanneman et al., 2006; Haslam and Dell, 2003). The most abundant class of glycans in C. elegans is the high-mannose type. This is followed by paucimannose and high-fucose type structures.

3. RNAi in C. elegans The genome sequence of C. elegans was first published in 1998 and contains approximately 20,000 genes (C. elegans Sequencing Consortium, 1998). One of the most useful features of C. elegans is its susceptibility to genetic silencing, most notably, RNA-mediated interference (RNAi). Doublestranded RNA that is complementary to a gene of interest can be easily directed through feeding, soaking, or injection and results in decreased gene translation through degradation of mRNA (Fire et al., 1998). The use of systematic reverse genetics has increased the capacity to investigate functional genomics and biological functions of glycosylation in C. elegans. Several genome-wide RNAi screens have been performed to study gene function in C. elegans. These RNAi are either derived from predicted genes in C. elegans, using information from the sequenced genome, or are made from cDNA, which represent genes that are expressed in laboratory conditions (Kamath et al., 2003; Rual et al., 2004). Genome-wide RNAi screens that used the NL2099 C. elegans strain, which is hypersensitive to RNAi, showed a 23% increase in the number of genes eliciting a phenotype compared to N2 Bristol (Simmer et al., 2003). This report highlighted the variability between genome-wide RNAi-by-feeding screens and calculated the figure to be 10–30%. It is important to note that neuronally expressed genes are relatively refractory to RNAi and may not be identified as tunicamycin-hypersensitive in our screen. Most RNAi experiments aim to understand the function of individual genes and to provide insight in the homologous human gene function. In our experiment, we targeted the synthetic interaction of each gene products’ loss-of-function with decreased LLO biosynthesis via tunicamycin treatment, thus providing a list of candidate genes that require N-glycosylation to function properly.

4. C. elegans Strains, Culturing and RNAi Methods C. elegans strains NL2099 rrf-3 (pk1426), VC569 tag-179 (ok809), RE666 ire-1 (v33), and N2 (Bristol) were obtained from the Caenorhabditis Genetics Center, University of Minnesota, USA. The VC569 strain, which originally contained a genetic balancer, was outcrossed with N2 (Bristol) to

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generate a viable strain that was ok809 homozygous. The balancer was removed in the fourth generation and was confirmed by polymerase chain reaction (PCR). General methods used for cultivating, handling, and genetic manipulation of C. elegans are as described (Brenner, 1974). All strains were grown on nematode growth media (NGM) containing 3 g NaCl, 3.5 g peptone, and 18 g agar in 1 l H2O. NGM were autoclaved at 121
C for 28 min, cooled to 65
C, and supplemented with 1 ml of 5 mg/ml cholesterol (in ethanol), 2 ml of 0.5 M CaCl2, 25 ml 1 M KPO4 at pH 6, and 1 ml MgSO4 (Brenner, 1974; Hope, 1999). Where RNAi was performed, NGM were supplemented with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for induction of double-stranded RNA and 50 mg/ml ampicillin (500 ml from 100 mg/ml stock) for RNAi plasmid selection. Following the additions, NGM were aseptically poured into 60 and 100 mm Petri plates or 24-well cell culture plates (Nunc). For tunicamycin treatment, NGM were supplemented with concentrations ranging 0–10 mg/ml. Tunicamycin (Calbiochem) was dissolved in dimethyl sulfoxide (DMSO) to 50 mg/ml stock concentration. C. elegans strains were fed OP50 Escherichia coli or a specific E. coli strain HT115(DE3) RNAi-by-feeding clone containing a L4440 vector with the RNAi target gene flanked by two T7 promoter regions. The T7 polymerase was induced by IPTG in the NGM, resulting in the expression of double-stranded RNA (dsRNA). RNAi screening protocols were performed by feeding in the NL2099 rrf-3 (pk1426) strain, with minor adaptations for tunicamycin treatment from previous works (Simmer et al., 2003). The ORFeome v1.1 RNAi library was supplied by the Vidal lab at the Dana-Farber Cancer Institute (Rual et al., 2004). The ORFeome v1.1 library of recombinant E. coli strains, consisting of 11,942 constructs (55% of the C. elegans genome), was arrayed in 96-well microtiter plates. The ORFeome library was stored at 80
C. Bacteria were cultured in 2  YT containing 50 mg/ml ampicillin and 12.5 mg/ml tetracycline for 72 h at 22
C to ensure the presence of the L4440 plasmid (ampicillin selection) and the DE3 lysogen carrying the IPTG inducible T7 RNA polymerase (tetracycline selection). From these cultures and using a 96-plate sterile stainless steel replicator, 100 ml 2  YT supplemented with 50 mg/ml ampicillin was inoculated and grown overnight at 37
C. Fifteen microliters aliquots of the overnight culture was used to grow bacterial lawns on NGM in each well of a 24-well cluster plate supplemented with 50 mg/ml ampicillin, 1 mM IPTG, and 2 mg/ml tunicamycin. These cultures were grown at 22
C for 48 h to create a bacterial food source expressing double-stranded RNA. 2  YT/Amp/Tet was prepared as follows: 16 g tryptone, 10 g yeast extract, 5 g NaCl in 1 l distilled H2O, autoclaved at 121
C for 15 min. Once cooled to 65
C, 2  YT was supplemented with 50 ml/ml ampicillin (500 ml from 100 mg/ ml stock) and 12.5 ml/ml tetracycline (500 ml from 25 mg/ml stock). Observations were made using a Leica MS 5 stereomicroscope at 10 to

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40 magnification (Leica Microsystems). All recognizable C. elegans phenotypes were scored using a controlled vocabulary, which are accessible via Wormbase, an online bioinformatic database resource for C. elegans genetics and biology (http://www.wormbase.org).

4.1. Effect of tunicamycin on C. elegans phenotypes The first step in the genome-wide RNAi screen was to characterize the developmental consequences of increased tunicamycin treatment (i.e., CDG-I-like defects) in C. elegans. To determine the effect of tunicamycin on C. elegans phenotypes, 15 ml of synchronized L1 stage N2 Bristol larvae following egg preparation were added to 60 mm NGM plates seeded with OP50 E. coli and supplemented with 0, 3, and 5 mg/ml tunicamycin. Animals were cultivated at 20
C and phenotypes were scored after 3 days. To obtain synchronous populations of L1 hatchlings, eggs were acquired by digesting populations containing gravid hermaphrodites with an alkaline hypochlorite mixture. Egg preparation method was as follows: NGM plates containing gravid hermaphrodites were washed with 5 ml 1  M9 and pelleted. A mixture of 250 ml NaOH (10 M) and 1 ml alkaline hypochlorite was added to the pellet and vortexed every 2 min for 10 min. The lysate was pelleted, supernatant was removed, and the pellet was resuspended in 5 ml 1  M9. The 1  M9 wash was repeated three additional times until the NaOH/hypochlorite solution was sufficiently removed. C. elegans eggs were allowed to hatch overnight in 1  M9 buffer at 20
C. 1  M9 buffer was prepared as follows: 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml MgSO4 (1 M) were autoclaved in 1 l H2O.

4.2. Tunicamycin-lethality is dose dependent To determine the lethal dose of tunicamycin during C. elegans larval development, synchronized L1 populations of N2 Bristol (n ¼ 144) following egg prep were placed in each well of a 24-well cell culture plate containing 2 ml NGM supplemented with tunicamycin (0–10 mg/ml) and seeded with OP50 E. coli. On days 3 and 6, plates were scored for death (no pumping, twitching, or movement when prodded with platinum wire) of the founder hermaphrodite or the appearance of hatched The number of animals that reached fertile adulthood at 20
C was scored. The percent of fertile live adults was plotted against the concentration of tunicamycin (Fig. 22.1).

4.3. Tunicamycin treatment varies among strains Similarly to the tunicamycin-lethality assay of N2 Bristol, separate populations of NL2099 rrf-3 (pk1426), RE666 ire-1 (v33), VC569 tag-179 (ok809), and N2 (Bristol) were tested in increasing concentrations of tunicamycin.

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Fraction of fertile adults

1.00

Day 3 Day 6

0.75

0.50

0.25

0.00 0

1

2

3 4 5 6 7 Tuicamycin (mg/ml)

8

9

10

Figure 22.1 Tunicamycin concentrations greater than 2 mg/ml induce a dose-dependent lethality during larval development in N2 Bristol strains. Each line represents the same observable population, but at days 3 and 6, respectively. Concentrations of eight or greater result in nearly complete lethality. Results are based on triplicate experiments (n ¼ 264) for each tunicamycin treatment. (Modified, with permission, from Struwe et al., 2009).

Each strain was synchronized by egg prep method as described above. Five microliters of hatchings (150 L1 larvae) in 1  M9 buffer was placed on 35 mm petri dishes containing 4 ml NGM with 0–10 mg/ml tunicamycin seeded with E. coli OP50. Animal survival was scored based on the ability of larvae to reach gravid hermaphrodites after 3 days at 20
C. The percent of gravid hermaphrodites (i.e., fertile adults) was plotted against the log scale concentration of tunicamycin (Fig. 22.2).

4.4. Establishment of tunicamycin-hypersensitive genes Table 22.1 and Figs. 22.1 and 22.2 illustrate that increased tunicamycin concentrations in NGM induce multiple phenotypes and lethality in C. elegans, as observed in CDG conditions. The key to the genome-wide screen is the ability to detect tunicamycin-hypersensitive genes, which is conditional on inducing a subphenotypic dose prior to RNAi knockdown. Otherwise, all detectable phenotypes and corresponding genes would be false positives from tunicamycin alone. Loci were scored positive if RNAi treatment in the presence of tunicamycin reproducibly caused a synthetic phenotype or unambiguously enhanced the phenotype observed in the absence of drug. From the aforementioned experiments, 2 mg/ml tunicamycin was chosen to screen a panel of 12 genes that were suspected to be tunicamycin-hypersensitive to validate the conceptual framework of the genome-wide screen. These included genes in LLO synthesis, ER-associated degradative pathway (ERAD), the unfolded protein response (UPR),

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120

% fertile adults

100 80 60 40 20 0 –2.0

–1.5

–1.0 – 0.5 0.0 0.5 (Tunicamycin) log (mg/ml)

1.0

NL2099 rrf-3(pk1426)II

VC569 tag-179(ok809)I

RE666 ire-1(v33)II

N2 (Bristol)

Figure 22.2 Tunicamycin affects postembryonic development among C. elegans strains. The percent of animals that reach fertile adulthood was measured as a function of tunicamycin concentration. Development of RE666 ire-1 (v33), which lacks a component of the unfolded protein response, is moderately affected when tunicamycin is present. Lethality is significantly increased in VC569 tag-179 (ok809), which encodes the enzyme responsible for the final step of LLO biosynthesis. Conversely, N2 (Bristol) and NL2099 rrf-3 (pk1426) strains are less affected by the presence of tunicamycin and are viable at 2 mg/ml. (Reproduced, with permission, from Struwe et al., 2009). Table 22.1 Tunicamycin treatment results in an increase in observable aberrant phenotypes [Tunicamycin] (mg/ml) Stage

Phenotype

0

3

5

Adult

W.T. Dpy Unc Sma Vab Egl Clr Dpy Unc Dpy Egl Gro Dpy Gro Let

99.81% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.05% 0.14% 0.00% 0.00% 2119

75.63% 3.39% 2.35% 2.04% 0.63% 0.57% 0.05% 0.05% 0.05% 1.51% 1.10% 9.71% 1916

1.01% 1.72% 0.20% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 14.34% 7.95% 74.77% 1974

Larval

n

Tunicamycin concentration at 3 mg/ml results in a wide variety of phenotypes among N2 adults and larvae. Concentrations at 5 mg/ml result predominantly in lethality. Clr, clear patches; Dpy, dumpy; Egl, egg-laying defective; Gro, slow growth; Let, lethality; Sma, small; Unc, uncoordinated locomotion; Vab, variably abnormal morphology; W.T., wild type. (Reproduced, with permission, from Struwe et al., 2009).

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Table 22.2 RNAi of genes involved in N-glycosylation with and without 2 mg/ml tunicamycin Function

Gene

Wormbase

 Tunicamycin

þ Tunicamycin

ERAD UPR UPR GVT GVT GVT LLO LLO LLO LLO LLO LLO

K08E3.7 C41C4.4 F46C3.1 W02D7.7 Y60A3A.9 F47G9.1 K09E4.2 C14A4.3 ZC513.5 C08B11.8 C08H9.3 T24D1.4 Vector

W.T. W.T. W.T. Gro, Unc, Pvl not tested not tested W.T. W.T. not tested W.T. W.T. W.T.

W.T. W.T. W.T. W.T. W.T. W.T. Gro W.T. W.T. W.T. W.T. W.T. W.T.

Ste, Emb, Gro Emb, Lva Gro Ste, Gro Let, Gro W.T. Emb Ste, Emb Emb, Lvl, Lva Gro Emb, Lvl, Lva Brd, Gro W.T.

RNAi of loci in the lipid-linked oligosaccharide (LLO) pathway, ER-associated degradative pathway (ERAD), unfolded protein response (UPR), or components of Golgi vesicle transport (GVT) presents no overt phenotype on NGM alone. In the presence of 2 mg/ml tunicamycin, RNAi knockdown of these genes generates multiple phenotypes. Corresponding RNAi phenotype experiments from Wormbase (http://www.wormbase.org) were are also shown for comparison. (Modified, with permission, from Struwe et al., 2009.)

and Golgi vesicular transport (GVT) (Table 22.2). Large scale RNAi was carried out on 24-well plates, where four wells containing NGM, 1 mM IPTG, and 50 mg/ml ampicillin with and without 2 mg/ml tunicamycin were inoculated with a specific RNAi expressing bacteria. Individual 24well plates contained six RNAi clones, each having four individual wells. In this manner, each gene tested provides four observable wells. Initially gravid hermaphrodite populations were synchronized via egg prep and allowed to hatch overnight at 20
C after transfer to NGM. Following egg prep, 10 NL2099 rrf-3 (pk1426) L3 animals were placed in the top row of each plate by hand using a ‘‘worm pick’’ of flattened platinum wire to generate progeny that have only existed in the presence of each RNAi construct with and without 2 mg/ml tunicamycin. After 48 h of ‘‘priming’’ at 15
C, a single gravid hermaphrodite from the top row was transferred to each of the other lawns and allowed to elaborate F1 progeny at 22
C for 72 h. Phenotypes in the four wells, including Po and F1 progeny, were scored. RNAi bacterium culture was prepared as follows: ORFeome v1.1 RNAi bacterial library was provided in 96-well microtiter plates, each well containing a specific bacterial clone. Each bacteria well was inoculated into 60 ml 2  YT/Amp/Tet in 96-well plate format using a sterile steel replicator. The inoculation plate was stored at 37
C for 18 h. Fifteen microliters aliquots of the overnight culture was used to grow bacterial lawns on

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24-well clusters. RNAi bacterial cultures were grown on NGM at room temperature for 48 h to create a bacterial food source expressing doublestranded RNA. This 24-well plate RNAi þ/ tunicamycin protocol was identical for the genome-wide screen. Our results were also compared to previously described RNAi phenotypes with an rrf-3(pk1426) mutant background of the same genes annotated in Wormbase (Kamath et al., 2003; Rual et al., 2004). The majority of RNAi clones tested correlated with previous works except in the case of sequence name C08H9.3, W02D7.7, and K09E4.2. Most important, this experiment established that tunicamycin-hypersensitive genes can be detected via RNAi in the presence of 2 mg/ml tunicamycin.

4.5. Tunicamycin does not alter RNAi effectiveness Our genome-wide screen was modeled similar to published RNAi screens in C. elegans (Simmer et al., 2003) with the exception that our NGM contained tunicamycin. Therefore the possibility that 2 mg/ml tunicamycin causes a generalized increase in the effectiveness of the RNAi-by-feeding method could not be excluded. If this was the case, then our approach would result in numerous false positives. To exclude this possibility, seven loci known to be refractory to RNAi (Asikainen et al., 2005) and that have observable mutant phenotypes were tested on 2 mg/ml tunicamycin correspondingly to the 24-well plate format described above. Briefly, bacterial constructs with RNAi targets of the seven genes were inoculated in 60 ml 2  YT with 50 mg/ml ampicillin and 12.5 mg/ml tetracycline for 72 h at 22
C. From these cultures, 100 ml 2  YT with 50 mg/ml ampicillin were inoculated and incubated for 18 h at 37
C. To each well of the 24-well plate, 20 ml of the culture was added to the surface of NGM containing 1 mM IPTG and 50 mg/ml ampicillin. Bacterial lawns were allowed to form, which expressed dsRNA specific to the seven genes tested. The NL2099 rrf-3 strain was used for the experiment as illustrated above. These results proved the legitimacy of the screen showing that tunicamycin does not elicit any phenotype from otherwise wild-type RNAi gene targets as previously reported by escalating the effect of dsRNA in C. elegans (Table 22.3).

5. Genome-wide RNAi Screen The first phase of the genome-wide RNAi screen was testing every gene from the ORFeome v1.1 library on tunicamycin with all phenotypes detected in any of the four wells being scored and cataloged. The type of phenotype was recorded and characterized as viable postembryonic phenotype (Vpep) or a nonviable phenotype (Nonv) as described (Simmer et al., 2003). The phenotypes of adults and progeny of the 10 founder NL2099 rrf-3

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Table 22.3 Tunicamycin treatment does not alter RNAi effectiveness in C. elegans Gene

Locus

Mutant

Wormbase (RNAi)

þ Tunicamycin

F11C3.2 W03A3.1 C52B9.9 C17D12.2 C52E12.3 D2096.4 ZC64.3

unc-122 ceh-10 mec-18 unc-75 spv-7 spv-1 ceh-18

UNC UNC UNC UNC LET LET LET

W.T. W.T. W.T. W.T. W.T. W.T. W.T.

W.T. W.T. W.T. W.T. W.T. W.T. W.T.

Seven C. elegans strains that exhibit both a mutant knockout phenotype and wild-type RNAi phenotype were chosen to compare RNAi in the presence of 2 mg/ml tunicamycin. No phenotype was detected when tunicamycin was present, demonstrating that tunicamycin does not modify the efficiency of RNAi. (Modified, with permission, from Struwe et al., 2009.)

animals (Po) in the ‘‘priming’’ well were scored in addition to the progeny and single founder hermaphrodite in wells 1–3. The genome-wide screen required strict criteria for candidate selection for the second phase, RNAi with and without 2 mg/ml tunicamycin, to limit the accumulation of false positives. This required determining how many wells that displayed phenotypes were required to be retested in the presence and absence of tunicamycin. To establish this threshold, a set of 100 genes that gave rise to phenotypes in 0–4 observatory wells out of four were tested in the presence and absence of 2 mg/ ml tunicamycin (n ¼ 500 total genes tested). As before, any animal in each well that showed a phenotype was scored as positive. In addition to the presence of a phenotype on tunicamycin (penetrance), the significance of the phenotype (either Vpep or Nonv) was assessed. The penetrance was assessed by subtracting the number of wells showing any phenotype(s) with tunicamycin from the number in its absence (D). The greater the D value, the more dependent the phenotype penetrance is on tunicamycin; that is, a locus that induced phenotypes in all four wells containing tunicamycin but none in its absence would have a D equal to 4. The effect of expressivity was assessed similarly except D was determined using the number of wells showing Nonv. For expressivity a larger D value corresponded to a more severe phenotype when tunicamycin is present; that is, a locus that induced a ‘‘dumpy’’ phenotype in all four wells in the absence of tunicamycin but ‘‘larval arrest’’ in all wells with 2 mg/ml would have D ¼ 4. Under this analysis, false positives and loci causing single-gene phenotypes score D ¼ 0, but loci that are tunicamycin-hypersensitive score D > 0. This analysis showed that when phenotypes were present in two or more wells out of four, the distribution was nonrandom. In phase II, any gene with two positive wells was retested with and without 2 mg/ml tunicamycin. The D values for penetrance and expressivity were plotted against the percent total in each of the five sets (100 genes per set) tested (Fig. 22.3).

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Expressivity

Number of phase I positive wells 4 3 2 1 0

80

% of class

60 40 20 0 –4

–3

–2

–1

0 Δ

Penetrance

2

3

4

Number of phase I positive wells 4 3 2 1 0

60 % of class

1

40 20 0 –4

–3

–2

–1

0 Δ

1

2

3

4

Figure 22.3 D values for expressivity and penetrance were calculated by subtracting the number of wells showing any phenotype with tunicamycin from the number in its absence. Larger D values correspond to a greater dependence of the phenotype penetrance/expressivity on tunicamycin. Phenotypes in  2 of 4 possible observable wells behave nonrandomly and were candidates for further testing in phase II ( 2 mg/ml tunicamycin). D values greater than two exhibit non-Gaussian distribution and indicate tunicamycin-hypersensitive loci. Sets of 100 genes that caused observable phenotypes in 0,1,2,3, and 4 observatory wells in the initial stages of the phase I screen were used for these assays (n ¼ 500 genes tested). (Modified, with permission, from Struwe et al., 2009).

For the genome-wide screen the preparation of media and bacterial cultures were followed as stated previously. All RNAi experiments were performed on the 24-well plate format supplemented with and without 2 mg/ml tunicamycin where indicated. There were two phases of the genome-wide screen: (1) screening the ORFeome v1.1 RNAi library in the presence of 2 mg/ml tunicamycin and (2) retesting candidates from phase I with and without 2 mg/ml tunicamycin to isolate tunicamycin-hypersensitive genes. Originally the two-phase scheme was chosen for cost considerations; however, the number of phase I candidates was higher than anticipated. Encouragingly, this resulted in an additional RNAi treatment

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results (screening on 2 mg/ml tunicamycin) which ultimately reinforced the validity of the 512 tunicamycin-hypersensitive genes discovered. During phase I, genes where at least two of the three replicates displayed concordant phenotypes were regarded as candidates. Any phenotypes observed in the ‘‘priming’’ well were also scored and data collected were weighed with lesser significance when selecting phase II candidates. The presence of any phenotypes in the three remaining wells, including Po and F1 progeny, were given a value of 10 (10 for any and all phenotypes and 0 for no phenotypes). Phenotypes in the ‘‘priming’’ wells were given a score of 5. In this manner, the evaluation of expressivity and penetrance of each gene tested can easily be calculated and interpreted. For example, the gene H23N18.4, which encodes a UDP-glucuronosyl and UDP-glucosyl transferase, had an expressivity score of 10 and a penetrance score of 30. In the absence of tunicamycin, all phenotypes were wild type (Vpep ¼ 0 and Nonv ¼ 0), but on 2 mg/ml tunicamycin, the phenotypes observed were slow growing (gro) in two wells and sterile (ste) in the third, thus the score for that gene was Vpep ¼ 30 and Nonv ¼ 10. Therefore the penetrance is 30 (30-0) and expressivity is 10 (10-0). These calculations helped to characterize the extent to which each gene contributed to the presence or severity of phenotypes observed during the phase II screen. Ultimately, 512 genes were deemed tunicamycin-hypersensitive and were annotated using the criteria above (Struwe et al., 2009). All recorded phenotypes were recorded initially in Microsoft Excel, and data were analyzed using SQL queries in Microsoft Access. Concise descriptions for each tunicamycin-hypersensitive gene product were added manually using Wormbase. Moreover, the tools Net-Nglyc (http://www.cbs.dtu.dk/services/NetNGlyc) and Proteome Analyst (http://path-a.cs.ualberta.ca) were helpful to predict N-glycosylation sites and the cellular location of each gene product, respectively. The most useful tool to annotate tunicamycin-hypersensitive gene function was the implementation of clusters of orthologous groups (COGs) functional classification, specifically sequenced genomes from eukaryotic orthologous groups (KOGs). KOG analysis was more effective than gene ontology descriptions, which proved to be either too broad or too specific when annotating the 512 tunicamycin-hypersensitive genes (Fig. 22.4).

6. Discussion There is an essential and extensive requirement for N-glycosylation in animal development. To understand the variable pathology of a CDG-Ilike condition, we developed an approach to systematically dissect the interactions between a primary etiologic defect (tunicamycin treatment) and modifier loci in the genetic background (tunicamycin-hypersensitive

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Translation, ribosomal structure and biogenesis Transcription Signal transduction mechanisms Secondary metabolites biosynthesis, transport and catabolism Replication, recombination and repair RNA processing and modification Posttranslational modification, protein turnover, chaperones Nucleotide transport and metabolism Nuclear structure Lipid transport and metabolism Intracellular trafficking, secretion, and vesicular transport Inorganic ion transport and metabolism General function predictions only Function unknown Extracellular structures Energy production and conversion Defense mechanisms Cytoskeleton Coenzyme transport and metabolism Chromatin structure and metabolism Call wall/membrane/envelope biogenesis Cell cycle control, cell division, chromosome partitioning Carbohydrate transport and metabolism Amino acid transport and metabolism

49 18 33 1 9 18 27 3 1 8 20 4 37 17 7 16 5 7 3 6 3 10 4 2 0

10

20

30

40

50

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Figure 22.4 Analysis of eukaryotic orthologous groups (KOGs) classify tunicamycinhypersensitive genes into functional sets. Of 512 genes found to be hypersensitive to tunicamycin, 308 had KOG assignments. (Reproduced, with permission, from Struwe et al., 2009).

genes) using RNAi. Our goal was to model the causes and consequences of CDG-I-like disorder in C. elegans, to perform a genetic analysis of N-glycosylation and the developmental mechanisms that depend on it. This genome-wide RNAi screen with ORFeome v1.1 library detected 512 tunicamycin-hypersensitive loci from 11,942 bacterial constructs. All RNAi screens have intrinsic limitations and for this reason we regard our approach as systemic rather than comprehensive. A major problem is that not all genes, particularly those expressed in neurons, are susceptible underscoring the need for complementary approaches such as the classical forward mutagenesis screen. Because high-throughput is necessary, only visible phenotypes were scored and it is virtually certain that several subtle traits are modulated by glycosylation that will be missed. However for our purpose, it is fortunate that RNAi generally reduces rather than eliminates expression; glycosylation cannot modulate the underlying polypeptide if it is absent, as would be the case in knockout models. Considering the C. elegans genome is roughly 20,000 genes, consideration should be given to additional RNAi screening (i.e., the remaining 8000 genes not tested in our initial screen). However, the advantage of the ORFeome RNAi library is that the RNAi constructs are generated from full coding region cDNAs; therefore the use of alternate RNAi

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libraries that are derived from genomic regions encompassing predicted genes may not prove more advantageous in detecting additional tunicamycin-hypersensitive genes. For that reason, future experiments should focus on screening any additional RNAi constructs from later versions of the ORFeome library. Moreover, additional work would aim to classify the 512 tunicamycin-hypersensitive genes according to the ‘‘maturation,’’ ‘‘etiologic,’’ and ‘‘effector’’ classes outlines in Fig. 22.5. Another consideration with the genome-wide RNAi screen is the use of tunicamycin. Tunicamycin treatment cannot be disregarded when considering the 512 tunicamycin-hypersensitive genes. The rational is that RNAi of a gene responsible for tunicamycin metabolism or uptake may alter the effect of 2 mg/ ml and result in nontunicamycin-hypersensitive gene identifications. Therefore, the 512 tunicamycin-hypersensitive genes should be retested with a knockout in the LLO pathway (preferably Y60A3A.14/T08D2.2: the UDPN-acetyl-glucosamine-1-P transferase) to effectively replace drug treatment. Here we outline a proof-positive approach that models a CDG-I-like disease and identifies loci that require N-glycosylation to function properly in C. elegans. This method could be applied to other organisms or tailored to model other diseases where modifier loci are difficult to resolve. This experiment is a powerful platform for investigating the molecular pathology of aberrant N-glycosylation. Our data demonstrates the first wide-ranging screen that successfully identified genes that require N-glycosylation to function and in doing so provides a list of candidate genes that may be screened to find enhancer loci in CDG-I patients and targeted for supportive treatment to minimize the severity of the illness.

Maturation genotypes

Effector genotypes

Trafficking

Underlying polypeptide genes Tunicamycin Dolichol

Lipid-linked oligosaccharide assembly

Monosaccharides

Oligosaccharyl -transferase

Nascent glycoprotein

Oligomer ER quality assembly control

Golgi Localization function Glycan remodeling

Mature glycoproteins

Phenotypes

Etiologic genotypes Unfolded protein response

Figure 22.5 Plausible interactions between tunicamycin and modifier loci in the genetic background in C. elegans. RNAi knockdown of ‘‘effector,’’ ‘‘etiologic,’’ or ‘‘maturation’’ genes interact with a decrease of LLO donor caused by 2 mg/ml tunicamycin treatment result in visible phenotypes. (Modified, with permission, from Struwe et al., 2009).

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