RNAi methodologies for the functional study of signaling molecules.

Abstract

RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules.

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RNAi Methodologies for the Functional Study of
Signaling Molecules
Gwang Lee1,2, Leah A. Santat3, Mi Sook Chang3, Sangdun Choi1,4*
1Department of Molecular Science and Technology, College of Natural Science, Ajou University, Suwon, Korea, 2Institute for Medical Sciences, College of Natural Science,
Ajou University, Suwon, Korea, 3Molecular Biology Laboratory, Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, California,
United States of America, 4Department of Biological Sciences, College of Natural Science, Ajou University, Suwon, Korea
Abstract
RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include
small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each
system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha
i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi)
platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then
developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of
target genes and shows the potential of RNAi for use in functional studies of signaling molecules.
Citation: Lee G, Santat LA, Chang MS, Choi S (2009) RNAi Methodologies for the Functional Study of Signaling Molecules. PLoS ONE 4(2): e4559. doi:10.1371/
journal.pone.0004559
Editor: Terry Means, Massachusetts General Hospital/Harvard University, United States of America
Received January 2, 2009; Accepted January 13, 2009; Published February 24, 2009
Copyright: ? 2009 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean Government (MOST) (R01-2007-000-
20533-0). This work was also partly supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2006-311-C00482) and
by the Ajou University Research Fellowship of 2008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: sangdunchoi@ajou.ac.kr
Introduction
RNA interference (RNAi) has been widely used by animal cell
biologists as a gene silencing tool to study the cellular effects
generated by modulating the expression of individual genes
[1,2,3,4,5,6]. Expression can be reduced by introducing gene-
specific double-stranded RNA (dsRNA) or single-stranded RNA
(ssRNA) into a cell. Subsequently, the small RNA products
generated from Drosha and/or Dicer-mediated dsRNA processing
are delivered to the RNA-induced silencing complex (RISC),
which is implicated in mRNA destruction and translational
repression, and the RNA-induced transcriptional silencing com-
plex (RITS), which is implicated in chromatin silencing [7,8,9].
However, difficulties remain in finding the best way to make
RNAi work as a gene-specific silencing procedure for signaling
studies. For example, dsRNA [10] or ssRNA [11,12,13] can
stimulate innate cytokine responses in mammals. Additionally,
synthetic small interfering RNAs (siRNAs) formulated in nonviral
delivery vehicles can be potent inducers of interferons and
inflammatory cytokines in mice and humans [14]. siRNAs can
also mediate sequence-independent gene suppression and induce
immune activation by signaling through toll-like receptor 3
(TLR3) [15]. A recent study showed that dsRNAs that are 21
nucleotides or longer are also involved in anti-angiogenic
responses [16]. These could significantly limit the application of
RNAi and result in off-target effects and immuno-stimulation
associated with the nucleic acid treatments. In order to develop
reliable RNAi reagents, three different platforms were tested: small
interfering RNA (siRNA), antisense oligonucleotide (ASO) and
short hairpin RNA (shRNA). siRNA, ASO and shRNA were
designed to knockdown the endogenous G protein alpha i2 (Gai2)
or G protein beta 2 (Gb2) gene in screens in RAW 264.7 murine
macrophage-like cells. A series of gene expression profiling
experiments and quantitative reverse transcriptase-polymerase
chain reaction (QRT-PCR) analyses were carried out to assess
both the specificity of the target gene knockdown and the
functional studies of the targeted signaling molecules. Gai2
knockdown cells were tested to monitor the changes in signaling
networks after treatment with different ligands, in this case
lipopolysaccharide (LPS), Pam2CSK4 (Pam2) or prostaglandin E2
(PGE2).
Results
DNA Microarray and RT-PCR verification of expression of
specific genes in RAW 264.7 cells
To develop a clear picture of the nature of the genes expressed
in RAW 264.7 cells, Affymetrix GeneChip screening was
performed, producing duplicate array data sets. Affymetrix Mouse
430 chips A and B were used to assess the gene expression of RAW
264.7 cells, mouse bone marrow derived macrophage (BMDM)
cells and the mouse macrophage cell line, J774A.1. BMDM and
the cell lines have ‘Present Call’ gene elements ranging from 17K
to 19K (data available at The Signaling Gateway: http://www.
signaling-gateway.org/data/micro/cgi-bin/micro.cgi?expt=affy).
Table 1 shows that a gene marked ‘Present’ in the Affymetrix
GeneChip data was almost always determined to be ‘Present’
when measured using RT-PCR. To avoid any possible errors in
the design of PCR primer sets, genes showing an ‘Absent’ signal in
RT-PCR during the first trial were verified using 2 to 3 alternative
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primer sets. Overall, approximately 5% of the transcripts found to
be present in the microarray were absent according to RT-PCR,
and 30–40% of absent determinations according to the microarray
were detected by RT-PCR (Table S1). However, in cases in which
multiple probes existed on the chip (38% of the probes),
reproducible calls (P: Present or A: Absent) improve the odds of
a correct result. The presence of Gai2 and Gb2 mRNAs was
verified using a DNA microarray and by a RT-PCR analysis
(Table 1).
Establishment of cells lacking Gai2 and Gb2 proteins
G protein coupled receptors are characterized by seven
transmembrane domains, and ligands stimulating these receptors
are diverse and include immuno-stimulating molecules. Hetero-
trimeric G proteins are composed of a, b and c subunits, with the
G protein alpha subunits consisting of four families: Gai/o, Gas,
Gaq/11 and Ga12/13. The Gai family is further characterized
into Gai1, Gai2 and Gai3. Gai2 has been reported to inhibit the
activities of types I, V, and VI adenylyl cyclase isoforms directly
[17]. As Gai2 appears to be involved in a complex pattern of
signaling
function was sought by silencing the Gai2 transcript. In addition,
the Gb2 subunits released upon activation of the heterotrimeric G
protein activate specific effectors, and previous studies have shown
it to be the primary beta subunit for certain Gai signaling
pathways in macrophage cells [5,6]. Thus, Gb2 was also targeted
to test the RNAi mechanisms.
RAW 264.7 cells were transfected with siRNA or ASO against
Gai2 or Gb2. Western blot results (Figures 1A and B) showed a
significant knockdown of the target gene in each of experiments.
RAW cells were transfected using FuGENE6 (Roche Molecular
Biochemicals, Indianapolis, IN) for ASO and Lipofectamine 2000
(Invitrogen, Carlsbad, CA) for siRNA. Cells were harvested at
72 hr post-transfection.
Pseudotyped lentivirus carrying shRNA can be efficiently
integrated into the chromosome, making it a good carrier for
the delivery and sustained expression of shRNA [5,28]. Lentiviral
vectors were constructed with expression cassettes that allowed the
expression of antibiotic selection markers (e.g., puromycin) in
order to identify and enrich the fraction of transduced cells
[17,18,19,20,21,22,23,24,25,26,27], insight into its
Table 1. Expression of heterotrimeric G protein subunits and RGS proteins in RAW 264.7 cells, as assessed by Affymetrix
GeneChips and RT-PCR (P: present, A: absent).
SymbolGene NameAffymetrix Calls RT-PCR ResultsAbundanceUMRR*
Gnai1 G alpha i1 subunitAAP
Gnai2 G alpha i2 subunitPP high
Gnai3G alpha i3 subunitPP medium
Gna11 G alpha 11 subunitPPmedium
Gna14 G alpha 14 subunitA,AAP
Gna15G alpha 15/16 subunitAP high
GnaqG alpha q subunitPPhigh
Gnb1 G beta 1 subunitP,A,PPhigh
Gnb2 G beta 2 subunitPP medium
Gnb3G beta 3 subunitAAP
Gnb4 G beta 4 subunitPPmedium
Gnb5G beta 5 subunit A,PPhigh
Gng2G gamma 2 subunitP,A,PP low
Gng3 G gamma 3 subunitAAP
Gng4G gamma 4 subunitA,A,AAA
Gng5G gamma 5 subunit P,P,AP medium
Gng7G gamma 7 subunitAP lowP
Gng8G gamma 8 subunitAP medium
Gng10 G gamma 10 subunitP
Gng11G gamma 11 subunitAPmedium
Gng12G gamma 12 subunitP
Gng13G gamma 13 subunitAAP
Gng14G gamma 14 subunitP
Rgs4regulator of G protein signaling 4A,A,AAP
Rgs7regulator of G protein signaling 7AAP
Rgs8regulator of G protein signaling 8P mediumP
Rgs13regulator of G protein signaling 13APlowP
Rgs16 regulator of G protein signaling 16A,A,AP high
Rgs19regulator of G protein signaling 19P,P PhighP
*RT-PCR results in Universal Mouse Reference RNA (11 cell line mixture).
doi:10.1371/journal.pone.0004559.t001
Gene Silencing
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(Figure 1C). Cells were infected with a lentivirus containing a
previously validated Gai2 or Gb2 shRNA [5,6]. The target gene
silencing activity of each shRNA in RAW264.7 cells was assessed
by QRT-PCR and immunoblotting for endogenous proteins.
Consistent with the QRT-PCR data, significant decreases in the
levels of Gai2 or Gb2 proteins were observed in the shRNA
expressing cells (Figure 1).
siRNA, ASO and shRNA as RNAi tools
It was confirmed by DNA microarray analysis using custom-
made inkjet-printed 16K oligonucleotide chips (Gene Expression
Omnibus platform accession number GPL254: http://www.
ncbi.nlm.nih.gov/projects/geo/) that the transfection of siRNA
against Gai2 or Gb2 showed significant levels of the target gene
knockdown (Figure 2). Cells were harvested at 48 hr post-
transfection for assessment of mRNA, and gene expression was
assessed using a DNA microarray and by QRT-PCR. Control
samples were mock-treated with transfection reagent to deter-
mine the baseline levels of gene expression. It was found that
400 nM siRNA is invariably more effective than 100 nM or
200 nM. Although several papers have noted that siRNA
reaches maximal effectiveness at a concentration lower than
100 nM [29,30], Song et al. [31] reported that primary
macrophages required 1 mM siRNA to achieve a maximal
effect. For any given siRNA, a comparable level of knockdown
can be achieved with at least a 10-fold lower siRNA
concentration using NIH3T3 cells (data not shown). The present
results suggest that the requirement for a high siRNA
Figure 1. Western blot analysis of (A) Gai2 and (B) Gb2 proteins in shRNA, siRNA and ASO treated RAW 264.7 cells. Two independent
experiments are shown for the siRNA and ASO treatments. Blots from the shRNA-expressing lentiviral (LV) lines are representative of multiple samples
taken over a 5-week period. Significant target gene knockdown is observed with all three platforms. UGIP: control lentivirus transfected cell line, UT:
untreated, Mock: mock-treated. (C) Schematic diagram of the lentiviral construct used to generate shRNA expressing RAW 264.7 cell lines. The hairpin
form of siRNA is expressed under the control of a mouse H1RNA polymerase III promoter. The vector also contains the enhanced GFP marker gene
and the puromycin resistance gene (Puro) regulated by a UbiC promoter. IRES, internal ribosome entry site; FLAP, HIV-1 FLAP element; WRE,
woodchuck hepatitis post- transcriptional regulatory element.
doi:10.1371/journal.pone.0004559.g001
Gene Silencing
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concentration in RAW264.7 cells is simply a function of their
low transfection efficiency.
Validated ASOs against the Gai2 or Gb2 gene were obtained
from ISIS Pharmaceuticals, Inc. (Carlsbad, CA). The ASOs used
were phosphorothioate oligodeoxynucleotides with 29-O-methox-
yethyl incorporated to enhance their affinity for RNA sequences
and their resistance to degradation by nucleases. RAW 264.7 cells
were transfected with 400 nM of ASO and were harvested for
mRNA analysis at 48 hr post-transfection. Results from the DNA
microarrays showed a detectable knockdown for ASO against Gb2
message (Figure 2B) as well as a specific knockdown for ASO
targeting Gai2. Another set of Gai2 ASO data was not included in
the analysis shown in Figure 2A due to the unexpected induction
of TLR-related genes. This will be discussed later.
It has been shown that the knockdown effect mediated by
siRNA or ASO is sustained for up to several days [29,32,33].
However, creation of a cell line infected with a shRNA-expressing
lentivirus provides an approach for gene knockdown over a longer
term. The analysis in Figure 2 shows that lentiviral vector-based
RNAi works well in RAW 264.7 cells with a robust knockdown of
the Gai2 and Gb2 target genes.
Induction of TLR-related genes by ASO designed for a
knockdown of Gai2
The ASO tests showed an unexpected observation of a
significant degree of induction of TLR-related genes by one of
ASOs designed for the knockdown of the Gai2 gene. Kim et al.
[34] found a very potent induction of interferon a and b by short
single-stranded RNAs (ssRNAs) transcribed with T3, T7 and
Sp6 RNA polymerases. In their studies, analyses of the potential
mediators of this response revealed that the initiating 59
triphosphate is required for interferon induction. Moreover,
single-strand RNA bearing 59 phosphate could activate RIG-1
mediated anti-viral responses [35] and it was also shown that 59
triphosphate is the ligand for RIG-1 [36]. However, with the
present ASO, neither RNA polymerase nor 59 triphosphate was
used. Nonetheless, a transcription profiling analysis revealed
patterns of regulation that were very similar to the patterns of
expression induced by ligands that trigger TLRs and clustered
with them (Figure 3), showing a higher expression of IL1b,
IFNa-inducible protein, chemokine (C-C motif) ligand 2 (Ccl2),
IFN-induced protein with tetratricopeptide repeats 1 (Ifit1), 2
(Ifit2) and 3 (Ifit3), granulocyte colony stimulating factor 3 (Csf3),
immunoresponsive gene 1 (Irg1), IL6, guanylate nucleotide
binding protein 1 (Gbp1), 2 (Gbp2) and 4 (Gbp4), dual specificity
phosphatase 1 (Dusp1) and 2 (Dusp2), IL10, and growth arrest
and DNA-damage-inducible 45a (Gadd45a) (not all data shown:
separate paper in preparation). When the ASO sequence for
Gai2 was studied further, it was found that the Gai2 antisense
oligonucleotide sequence has several CpG motifs (59-TTT AGA
GCG CTC GGC TGC CG-39: multiple unmethylated CpG
appeared and one purine-purine-CpG-pyrimidine-pyrimidine
existed). Unmethylated CpG motifs are present in bacterial
genomic DNA and function as a pattern recognition motif by the
host innate immune system [37,38]. For mouse, the sequence
GACGTT appears to be optimal, but flanking nucleotides to the
Figure 2. Hierarchically clustered dendrograms of gene expression changes. Clustering was achieved using the CLUSTER and TREEVIEW
programs (http://rana.lbl.gov/EisenSoftware.htm). Each row represents a gene, and each column represents a particular sample. (A) Gai2 gene
knockdown (LV40 and LV11: shRNA- expressing lentivirus transfected cell lines; I and L: I or L form of siRNA transfected into cells). (B) Gb2 gene
knockdown (LV4 and LV43: shRNA-expressing lentivirus transfected cell lines; G and H: G or H form of siRNA transfected into cells; Gb2-ASO: antisense
oligonucleotide). The expression level relative to that of the control cells is provided by colors shown in log2scale. The target genes, Gai2 and Gb2,
are highlighted by arrows. The apparent down-regulation of IL13ra2 in the Gai2-deficient cells and IL1rn in the Gb2-deficient cells are also highlighted
(see context).
doi:10.1371/journal.pone.0004559.g002
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central CpG core appear to influence recognition to some
degree. Oligonucleotides containing the core CpG motif might
bind to the TLR9 whose expression was confirmed in RAW
264.7 cells (Table 2). TLR9 engagement triggers alteration of the
cellular redox balance, tyrosine phosphorylation of vav1 by a src-
related tyrosine kinase [39], and the induction of cell signaling
pathways including mitogen-activated protein kinases (MAPKs)
and NFkB [37]. Interleukin-1 receptor-associated kinase (IRAK)
and/ortumor-necrosis-factor-receptor-associated
(TRAF6) may be diverging points for NFkB activation in
response to CpG DNA in RAW264.7 cells [40,41]. In our ASO
sequence against Gai2, three copies of the unmethylated CpG
motif per oligonucleotide led to the enhanced activation of
TLRs.
factor6
Figure 3. Hierarchical clustering of gene expression for different RNAi platforms or ligand treatments in Gai2 knockdown RAW
264.7 cells. While Pam20.5 h (1st) and Pam20.5 h (2nd) were tested in lentivirus vector transfected RAW cell lines, Pam20.5 h was carried out in
wild type RAW 264.7 cells together with other TLR ligand experiments, in this case Pam3, Zymosan, Taxol, PolyIC and CpG.
doi:10.1371/journal.pone.0004559.g003
Gene Silencing
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