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From: Mikiko C. Siomi, Kazumichi M. Nishida, and Haruhiko Siomi,
How to Define Targets for Small Guide RNAs in RNA Silencing:
A Biochemical Approach
In Lynne E. Maquat and Megerditch Kiledjian, editors,
Methods in Enzymology, Vol 449,
Burlington: Academic Press, 2008, pp.345-355.
ISBN: 978-0-12-374584-2© Copyright 2008 Elsevier Inc.
Academic Press.

Author's personal copy

C H A P T E R

S I X T E E N

How to Define Targets for
Small Guide RNAs in RNA Silencing:
A Biochemical Approach
Mikiko C. Siomi,*,† Kazumichi M. Nishida,*,‡ and Haruhiko Siomi*
Contents
1. Introduction
2. Immunopurification of Aub–piRNA Complexes from
Fly Testis Lysates
3. Analyzing Protein Components Present in Immunoprecipitates by
Silver Staining and Western Blot Analysis
4. Analyzing Small RNAs Present in Immunoprecipitates by
Northern Blot Analysis
5. Target RNAs for Small RNA-Guided Cleavage
6. In Vitro Target RNA Cleavage (Slicer) Assay
Acknowledgments
References

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Abstract
RNA silencing involves various forms of sequence-specific gene silencing triggered by small RNAs. In RNA silencing, Argonautes are crucial protein components that are directed to the target messenger RNAs (mRNAs) through theirassociation with small RNAs by base pairing. Argonautes repress the expressionof the target genes at posttranscriptional levels. Full complementarity between
a small RNA and its target mRNA results in Argonaute-mediated cleavage(‘‘slicing’’) of the target mRNA. The D-D-H (asparagine-asparagine-histidine)
triad that exists in the PIWI domain of Argonautes is the catalytic center for
rendering their target cleavage (‘‘slicer’’) activity. This chapter describes in vitro
target RNA cleavage assays using Aubergine in a complex form with PIWIinteracting RNAs. Aubergine is one of the Argonautes expressed primarily in
fly germ lines and is immunopurified from fly testes using the specific antibody
against it. The method discussed is useful for defining targets for the small
RNAs that function in RNA silencing.
* Keio University School of Medicine, Tokyo, Japan{{

JST, CREST, Saitama, Japan
Institute for Genome Research, University of Tokushima, Tokushima, Japan

Methods in Enzymology, Volume 449
ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)02416-6# 2008 Elsevier Inc.

All rights reserved.

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1. Introduction
RNA silencing is an evolutionally conserved cellular process in whichsmall RNAs trigger various forms of sequence-specific gene silencing(Tomari and Zamore, 2005; Zaratiegui et al., 2007). In each case, thesmall RNAs act as guide molecules that direct effecter complexes to target
nucleic acids by base pairing.
RNA interference (RNAi) (Fire et al., 1998) is the prototype of RNA
silencing and causes gene silencing by cleaving (slicing) the target mRNAs ina sequence-dependent manner (Tomari and Zamore, 2005). The sequencespecificity is rendered by a 21-to-23-nucleotide RNA, termed short interfering RNA (siRNA) (Tomari and Zamore, 2005). Micro-RNAs (miRNAs) are a large subset of endogenous small RNAs (21–23 nucleotides inlength) encoded in the genome of a variety of organisms (Ambros, 2004;
Kloosterman and Plasterk, 2006; Siomi and Siomi, 2007). miRNAs alsofunction in RNA silencing, where the expression of genes involved invarious developmental and metabolic processes is repressed posttranscriptionally. The origin of siRNAs and miRNAs differ from one another but
both are processed from a longer double-stranded RNA precursor by an
RNase III domain-containing Dicer protein and loaded onto a member of
the argonaute family of proteins (Carmell et al., 2002; Parker and Barford,2006).
Members of the Argonaute family are defined by the presence of the
PAZ and PIWI domains (Carmell et al., 2002). In Drosophila, five genes,
AGO1, AGO2, AGO3, Piwi, and Aubergine (Aub), are encoded as membersof the Argonaute family and, according to sequence, are divided into two
subfamilies, namely AGO and PIWI. AGO1 and AGO2, members of the
AGO subfamily, are expressed ubiquitously through development. The
other three, members of the PIWI subfamily, are only expressed in germline cells (Williams and Rubin, 2002).
AGO1 and AGO2 in Drosophila function in gene silencing throughspecific binding with miRNA and siRNA, respectively (Miyoshi et al.,2005; Okamura et al., 2004; Tomari et al., 2004). AGO2 functions in
RNAi as Slicer and is directly responsible for cleaving a target mRNA
that is completely complementary to the siRNA (Miyoshi et al., 2005).
Although AGO1 is thought to function in miRNA-mediated translational
repression, it also possesses Slicer activity (Miyoshi et al., 2005). However,
why and when AGO1 acts as a Slicer remains unelucidated.
Studies have shown that Piwi, Aub, and AGO3, PIWI subfamily members in Drosophila, are associated with a subset of endogenous small RNAs,
termed piRNAs (PIWI-interacting small RNAs; Brennecke et al., 2007;
Gunawardane et al., 2007; Nishida et al., 2007; Saito et al., 2006; Vagin et al.,2006). piRNAs were originally found in the small RNA profiling studies inAuthor's personal copy
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Drosophila testis and embryo (Nishida et al., 2007) as a subgroup of small
RNAs (24–30 nucleotides in length) that are distinct from miRNAs. (In theinitial report, rasiRNA was the original term used for the small RNAs foundin germ lines/embryos; however, it was revealed more recently that rasiRNAs are a subset of piRNAs; thus, the term ‘‘rasiRNA’’ is not used in thischapter.) While miRNAs are processed by Dicer from hairpin-formed
precursors encoded in the genome, piRNAs are derived from a variety ofrepetitive intergenic elements such as retrotransposons that are embedded in
the genome and produced by a Dicer-independent pathway (Aravin et al.,
2003; Brennecke et al., 2007; Saito et al., 2006; Vagin et al., 2006). Proteinsof the PIWI subfamily produced in Escherichia coli and immunopurified fromgerm line cells are capable of exhibiting Slicer activity in vitro (Brennecke
et al., 2007; Gunawardane et al., 2007; Miyoshi et al., 2005). Taken together,
it was postulated that the PIWI proteins function, at least in part, as Slicer
in silencing repetitive/transposable genes through their association with
piRNAs and so protect the genome from invasive elements.
The most abundant (about 46% of the total) class of piRNAs associatedwith Aub in testes consists of those that derive from Suppressor of Stellate[Su(Ste)] antisense transcripts (Nishida et al., 2007). Su(Ste) genes are locatedrepeatedly on the Y chromosome, show a significant similarity to Stellate
genes at the nucleotide level, and are considered paralogs of Stellate genes(Livak, 1984). Although Su(Ste) genes produce nonprotein-coding transcripts, Stellate genes encode a protein with striking similarity to the b subunitof protein kinase CK2 (Livak, 1990) and are found repetitively on the X
chromosome (Livak, 1984). Deletion of the Su(Ste) repeats on the
Y chromosomes results in overexpression of Stellate in testes and causes anaccumulation of aggregates containing Stellate proteins in spermatocytes(Bozzetti et al., 1995). Aggregation then causes male infertility because of
severe defects in spermatogenesis. Thus, silencing of Stellate genes by Su(Ste)repeats is essential for the maintenance of male fertility. Genetic approaches
later revealed that mutations in the Aub gene cause male sterility, which canbe attributed directly to a failure to silence the repetitive Stellate locus(Schmidt et al., 1999). Biochemical and genetic studies showed that the Su(Ste) locus produces piRNAs and that the Aub gene is required for theaccumulation of Su(Ste) piRNAs (Aravin et al., 2004; Vagin et al., 2006).
However, a physical connection between Aub and Su(Ste) piRNAs has not
yet been determined and how Aub is involved in silencing the Stellate genes
remains unanswered. Through a profiling study for piRNAs associated with
Aub in testes, we found that Aub interacts physically with piRNAs derived
from Su(Ste) antisense transcripts in testes (Nishida et al., 2007). Although thenucleotide sequences of Stellate and Su(Ste) genes are not identical, they
are strikingly similar to each other. Moreover, we demonstrated that Aub
produced in E. coli shows Slicer activity in vitro (Gunawardane et al., 2007).
Considering the aforementioned findings, we speculated that Aub complexes

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containing Su(Ste) piRNA silence Stellate genes by cleaving the Stellate transcripts, analogous to how the AGO2–siRNA complexes function in RNAi
(Miyoshi et al., 2005). That is to say, Stellate could be one of the ‘‘targets of
Aub’’ in testes. To examine if it is indeed the case, the Aub–piRNA complexes
were immunopurified from the testis lysate and in vitro target RNA cleavage
assays were performed. We demonstrated that the Aub complexes immunopurified from testes were able to cleave a target RNA containing part of theStellate transcript (Nishida et al., 2007). Results indicated that Stellate genes area bona fide target of Aub and that Su(Ste) piRNAs can trigger silencing of
Stellate in concert with the Slicer activity of Aub in testes.
This chapter describes the details of (1) how Aub–piRNA complexes are
immunopurified from Drosophila testes as a model system, (2) how target
RNAs are prepared for in vitro target RNA cleavage assays, and (3) how
in vitro target RNA cleavage assays are performed. These methods will be
useful for identifying small guide RNAs, as well as defining targets for thesmall RNAs identified in any organisms.

2. Immunopurification of Aub–piRNA Complexes
from Fly Testis Lysates
Testes are dissected manually from adult flies in 1 phosphatebuffered saline (PBS). About 1000 testes are collected in a 1.7-ml microcentrifuge tube and washed with 1 ml of 1 PBS three times. Each time,
testes were spun briefly at 4  C. After removing and discarding as muchbuffer as possible, testis lysates are prepared by grinding them in a cold lysis
buffer-containing 30 mM HEPES-KOH (pH 7.3), 150 mM KOAc, 2 mM
MgOAc, 5 mM dithiothreitol (DTT), 0.1% NP-40, 2 mg/ml pepstatin,
2 mg/ml leupeptin, and 0.5% aprotinin. During this step, the microcentrifuge tube should be kept on ice. After spinning at 15,000 rpm for 1 min at
4  C, the supernatant is transferred to a new 1.7-ml microcentrifuge tube
and kept on ice. Lysis buffer is added again to the residual pellets and groundagain for reextraction. This step is repeated several times and all the supernatant is then gathered in a microcentrifuge tube. At this point, the final
volume should be about 0.5 ml. The supernatant is spun at 15,000 rpm for 20
min at 4  C to remove all unnecessary debris and is then transferred to a new
microcentrifuge tube. This supernatant is a testis lysate that can be used for
immunoprecipitation experiments. Immunoprecipitation from the supernatant is performed with the anti-Aub monoclonal antibody (Gunawardane et al.,2007) immobilized on Gamma Bind beads (GE Healthcare) (see Note 1).
The amount of anti-Aub antibody used is about 1 ml of the culture
supernatant from hybridoma cells generating the anti-Aub antibody. One
milliliter should give rise to about 10 mg of anti-Aub antibody. Alternatively,

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a purified antibody of the same amount can be used for this step. The lysate–
antibody mixture is then rocked at 4  C for at least 1 h and the beads are
washed extensively with 1 ml of the lysis buffer (kept on ice); this is done atleast five times. The final wash is carried out with a cleavage reaction buffer(30 mM HEPES-KOH, pH 7.4, 40 mM KOAc, 5 mM MgOAc, and 5 mM
DTT) (see Note 2).

3. Analyzing Protein Components Present in
Immunoprecipitates by Silver Staining andWestern Blot Analysis
After extensive washing of the beads, immunoprecipitated proteins are
eluted with SDS sample buffer without DTT (see Note 3). After elution,
DTT is added to a final volume of 100 mM. Samples are then boiled andloaded onto SDS-acrylamide gels. After electrophoresis, protein bands arevisualized by silver staining using SilverQuest (Invitrogen; Fig. 16.1A). To
ascertain if Aub is indeed contained in the immunoprecipitates, Western blot
analysis of the immunoprecipitates should be carried out using the anti-Aub
antibody (Fig. 16.1B). To do so, after electrophoresis, protein components aretransferred to a Protran nitrocellulose transfer membrane (Whatman) andprobed with the anti-Aub antibody for at least 1 h at room temperature.
Prior to this, the membrane should be incubated in blocking buffer tominimize nonspecific protein–antibody interactions with 1 PBS buffer
containing 5% nonfat milk for a half an hour at room temperature. Afterincubation with the primary antibody, the membrane is washed three times
with PBST buffer (0.1% Tween-20 containing 1 PBS) and incubated in
PBST buffer containing the secondary antibody conjugated with peroxidase(peroxidate-conjugated sheep IgG fraction to mouse IgG; MP Biomedicals). After extensive washing with PBST buffer, the proteinband corresponding to Aub can be visualized using detection reagent 1.2(GE Healthcare) according to the manufacturer’s instructions.

4. Analyzing Small RNAs Present in
Immunoprecipitates by
Northern Blot Analysis
RNAs that are bound by, and immunoprecipitated with, the Aubprotein from testes extract can be isolated using the ISOGEN RNA extraction
procedure according to the manufacturer (Nippon Gene). The resultant
RNAs are resolved in a 6 M urea-containing 12% acrylamide denaturing
gel and transferred to Hybond-Nþ (GE Healthcare) by electrophoresis.

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A

N.i.

Anti-aub

MW

IP fromtestisAnti-aub only

Mikiko C. Siomi et al.

220160120100--Aub

806050-(kDa)

Anti-aub

Input

N.i.

IP fromtestisB-Aub

Figure 16.1 Analyzing the protein components in Aub immunoprecipitates. (A)Silver-stained proteins immunoprecipitated from Drosophila testes using anti-Aub antibody. N.i., nonimmune IgG (negative control). In anti-Aub immunoprecipitates, a bandcorresponding toAub itself is clearly observed. (B) Western blot analysis of the immunoprecipitates shown in (A). Anti-Aub antibody was used as the primary antibody. N.i.,
nonimmune IgG (negative control).

After transferring, the membrane is UV cross-linked and baked at 80  C for
1 h to immobilize the RNAs. An oligonucleotide probe (a DNA oligonucleotide containing a sequence fully complementary to the small RNA to be
detected) is labeled with T4 polynucleotide kinase in the presence of [g-32P]
ATP and hybridized to the membrane at 42  C in 7% SDS containing 0.2 M
Na2PO4 (pH 7.9) overnight. The membrane is then washed twice at 42  Cwith 2 SSPE containing 0.1% SDS. The blot is exposed to an imaging
plate, and signals are analyzed on BAS-2500 (Fuji) or equivalent.

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5. Target RNAs for Small RNA-Guided Cleavage
To make a target harboring a partial sequence of Stellate sense mRNA
(Stellate target), a short DNA fragment produced by annealing a set of DNA
oligonuclotides (50 -AATTCTCTGGCTTGTTGTACGGCGATGAAAG30 and 50 -GATCCTTTCATCGCCGTACAACAAGCCAGAG-30 ) is
inserted into a pBS SKþ vector digested with BamHI and EcoRI. This
yields a plasmid containing the Stellate target sequence. The sequence ofStellate mRNA was selected as the target sequence because it shows striking
complementarity to Su(Ste)-4 piRNA, the most abundant piRNA found in
Aub–piRNA complexes in testes (Nishida et al., 2007). The Stellate target
sequence is polymerase chain reaction (PCR) amplified from the Stellate
target-containing plasmid using primers for the T7 and T3 promoter
sequences. The resulting PCR products are subsequently used as templates
for in vitro transcription using a MEGAscript T7 Kit (Ambion). Transcription is performed according to the manufacturer’s instructions. After thereaction, the transcribed RNA is electrophoresed in a 6 M urea-containing
6% acrylamide denaturing gel, which is then stained with toluidine blue O
solution (0.016% toluidine blue O) (Waldeck), 2% methanol, and 0.04% aceticacid), and the target RNA band with the expected size is excised from the gelusing a clean razor blade. The gel piece is put in a 1.7-ml microcentrifuge tube
and crushed into small pieces by grinding with a disposable pipette tip. Two gelvolumes of RNA elution buffer (0.5 M ammonium acetate, 1 mM EDTA,
0.2% SDS) are added to the tube and rotated at 4  C overnight to elutethe RNA fragments. The gel pieces are sedimented by centrifugation(at 15,000 rpm for 1 min), and the supernatant is transferred to a new
microcentrifuge tube. The target RNA is extracted using phenol and is
subsequently precipitated with isopropanol in the presence of Pellet-paintCo-precipitant (Novagen). The resulting RNAs are then radiolabeled attheir 50 ends by the addition of a 50 -m7G cap using guanylyltransferase(Ambion) (see Note 4). To do so, combine the following capping reaction
mixture in a total volume of 15 ml: 1.5 ml of 10 capping reaction buffer(Ambion), 4.5 ml of [a-32P]-labeled GTP (3000 Ci/mmol) (Perkin Elmer),
1 ml of S-adenosyl methionine (Ambion), 0.5 mg of gel-purified target
RNA, and 1 ml of guanylyltransferase enzyme. The reaction mixture is
incubated at 37  C for 60 min and filtered through a P-30 column, according to the manufacturer’s instructions (Bio-Rad), to remove unincorporated[a-32P]GTP. The target RNA labeled is again gel-purified from a 6%
acrylamide denaturing gel as indicated earlier. The resultant RNA is dissolvedin nuclease-free water to a working concentration of 3000–5000 cpm/ml.

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6. In Vitro Target RNA Cleavage (Slicer) Assay
Gamma Bind beads containing the immunopurified Aub protein fromtestes (see the earlier discussion) are drained and the following materials areadded to the beads.
 6 ml of 5 cleavage buffer (150 mM HEPES-KOH, pH 7.4, 500 mM

KOAc, 25 mM MgOAc, 25 mM DTT, 50 mM creatine phosphate, and
2.5 mM ATP)
 1 ml of RNasin plus (3 U/ml) (Promega) 1 ml of yeast RNA (0.5 mg/ml) (Ambion) 1 ml of creatine kinase (900 mg/ml) (Roche) 1 ml of target RNA (3000–5000 cpm/ml)
The final volume is adjusted to 30 ml by adding H2O. The reaction mixture
is incubated at 26  C for 180–270 min. The resultant RNA is then purified
using ISOGEN-LS (NipponGene) and separated in a 6% acrylamide denaturing gel. The gel is exposed to an imaging plate and the gel image isanalyzed using a BAS-2500 (Fuji; Fig. 16.2) or the equivalent.

Anti-AubN.i.

IP fromtestis- Target RNA- Cleavage
product
Expected cleavage site
Stellate target
3’-------aaaguagcggcauguuguucggucu-------5’
5’-ucucaucgucguagaacaagcccga-3’
Su(Ste)-4 piRNA

Figure 16.2 Aub^piRNA complexes show Slicer activity in vitro. An in vitro target
RNA cleavage assay was carried out using Aub complexes immunopurified from testes(Fig. 16.1). An RNA target containing part of the Stellate transcript was used. The
expected cleavage product is observed with the Aub complexes, but not in the negative
control lane (N.i.), indicating that Aub complexes from testes have the ability to cleave
the target RNA. N.i., nonimmune IgG (negative control). Sequences of Su(Ste)^4
piRNA and part of the Stellate transcript are shown in a box on the right.The expectedcleavage site on the target is indicated by an arrow.

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Notes
Note 1. From our experience, the volume of the Gamma Bind beads shouldbe about 20 ml. When the volume is more than 20 ml, the background isoften undesirably high.
Note 2. We previously immunopurified Ago1–miRNA complexes from
S2 cells under stringent conditions with a salt concentration of 1 M NaCl tostrip away associated proteins (in particular, other argonautes) (Miyoshi
et al., 2007). Under the same salt concentration, most of the proteins thatare associated with human Ago2 are also stripped away from the complexes
when immunopurified with specific monoclonal antibodies from humancells (HeLa; Miyoshi et al., 2008). However, we noticed that such stringent
conditions are not necessary in the case of immunopurifying Aub–piRNAcomplexes. Indeed, we confirmed by Western blot analyses that even undernearly physiological conditions (see earlier discussion) the Aub complexes
did not contain other argonaute proteins (Nishida et al., 2007). However, if
desired, the argonaute–small RNA complexes should be purified undermore stringent conditions. Indeed, it has been noted that the potassium
chloride concentration in the wash steps of the affinity column purification
could be increased up to 2.5 M without loss of RISC activity (Martinez andTuschl, 2004). It is critical to adjust immunoprecipitation conditions(including salt and/or detergent concentrations and binding times) empirically according to the focus of the particular experiment.
Note 3. At this stage, it is quite important to use SDS sample buffer
without DTT, as DTT elutes most antibodies from the beads and, as aresult, the background of the silver-stained gel becomes much higher.
Note 4. The particular RNA used in the in vitro target RNA cleavage
assays can be radiolabeled at the 50 end by guanylyltransferase in the presenceof [a-32P]GTP or by T4 polynucleotide kinase in the presence of [g-32P]ATP. Alternatively, the target RNA can be radiolabeled at the 30 ends by
RNA ligase in the presence of [32P]-pCp (Nishida et al., 2007).

ACKNOWLEDGMENTS
We thank Keita Miyoshi for comments and advice on the manuscript. This work was
supported by grants to M. C. Siomi and H. Siomi from Ministry of Education, Culture,
Sports, Science and Technology of Japan, and the New Energy and Industrial Technology
Development Organization. M. C. Siomi is also supported by CREST from JST. Mikiko C.
Siomi is Associate Professor of Global COE for Human Metabolomics Systems Biology by
MEXT.

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