RNA Helicase A Interacts with RISC
in Human Cells and Functions in RISC Loading
G. Brett Robb1and Tariq M. Rana1,*
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester,
MA 01605, USA
RNA interference is a conserved pathway of
sequence-specific gene silencing that depends
on small guide RNAs and the action of proteins
assembledin the RNA-induced
complex (RISC). Minimally, the action of RISC
requires the endonucleolytic slicer activity of
Argonaute2 (Ago2) directed to RNA targets
whose sequences are complementary to RISC-
incorporated small RNA. To identify RISC
components in human cells, we developed an
affinity-purification strategy to isolate siRNA-
programmed RISC. Here we report the identifi-
cation of RNA helicase A (RHA) as a human
RISC-associated factor. We show that RHA
interacts in human cells with siRNA, Ago2,
TRBP, and Dicer and functions in the RNAi
pathway. In RHA-depleted cells, RNAi was re-
duced as a consequence of decreased intracel-
lular concentration of active RISC assembled
with the guide-strand RNA and Ago2. Our re-
sults identify RHA as a RISC component and
demonstrate that RHA functions in RISC as an
RNA interference (RNAi), the sequence-specific silencing
of gene expression, is directed by small RNAs acting in
concert with several proteins in the RNA-induced silenc-
ing complex (RISC). Although human RISC activity can
be reconstituted in vitro using single-stranded siRNA
and recombinant Argonaute2 (Ago2) (Rivas et al., 2005),
in vitro formation of human RISC using double-stranded
RNAs requires additional factors such as Dicer and trans-
activation-responsive RNA binding protein (TRBP) (Chen-
drimada et al., 2005). RISC fractionates in large molecular
mass complexes containing multiple proteins, many of
which have been identified by immunoaffinity purification
of Ago2 from cell extracts. Some of these proteins appear
to play a functional role in RNAi. For example, depleting
MOV10 (Moloney leukemia virus 10, homolog) or TNRC6B
from cells prevented silencing of microRNA-targeted re-
porter gene expression (Meister et al., 2005). Knockdown
of Dicer, TRBP, or protein activator of the interferon-
induced protein kinase (PACT) reduced the siRNA-
induced silencing of the reporter gene (Chendrimada
et al., 2005; Haase et al., 2005; Lee et al., 2006). Thus,
while siRNA and Ago2 constitute the core endonucleolytic
components of the RISC, other proteins clearly modify
and enhance their function in gene silencing.
RHA (also known as DHX9 and NDHII) is a member of
the DEAH-containing family of RNA helicases (Abdelha-
leem et al., 2003). Originally purified and characterized in
HeLa cells (Lee and Hurwitz, 1992), human RHA was later
identified as the ortholog of Drosophila maleless protein
(Lee and Hurwitz, 1993). RHA enzymatically unwinds
RNA-RNA duplexes as well as RNA-DNA hybrids in a 30
to 50direction, provided that the RNA has unpaired 30
ture helicase core domain, RHA also contains three RNA
binding domains that include two N-terminal double-
stranded RNA binding domains and a C-terminal RGG
box domain, allowing RHA to bind double- and single-
stranded RNA as well as single-stranded DNA (Lee and
The Drosophila RHA ortholog, maleless, functions in
transcriptional upregulation of the male X chromosome
1998). Likewise, RHA plays many roles in transcriptional
regulation of human genes. RHA occupies the promoters
of the p16INK4a (Myohanen and Baylin, 2001) and the
MDR1 (Zhong and Safa, 2004) genes, from which it en-
hances transcription. In addition, RHA interacts directly
with the p65 subunit of nuclear factor-kB and stimulates
its transcriptional regulatory activity (Tetsuka et al.,
2004). RHA also acts as a bridging factor, coupling RNA
polymerase II (RNAPII) to the transcriptional coactivators
BRCA1 and CBP/p300 (Anderson et al., 1998; Nakajima
factor between the SMN (survival of motor neurons) com-
plex and RNAPII, linking RHA to splicing/cotranscriptional
mRNA processing (Pellizzoni et al., 2001).
RHA localizes to both the nucleus and the cytoplasm of
human cells and shuttles between the two compartments
by virtue of a bidirectional nuclear transport domain at the
protein’s C terminus (Tang et al., 1999). RHA binds the
constitutive transport element (CTE) of type D retroviruses
to facilitate expression of CTE-dependent genes and
Molecular Cell 26, 523–537, May 25, 2007 ª2007 Elsevier Inc. 523
plays a role in HIV-1 RRE-dependent gene expression (Li
et al., 1999; Tang et al., 1999). RHA was recently shown to
be required for the translation of JUND and a posttran-
scriptional control element-containing viral mRNA (Hart-
man etal.,2006).Translation of PCEmRNAwasproposed
to be stimulated by RHA remodeling of complex protein-
RNA 50untranslated region structures that inhibit efficient
ribosomal scanning of these transcripts.
Since RHA appears to regulate many aspects of gene
expression, it is not surprising that murine RHA is required
for development. RHA null embryos fail to undergo normal
gastrulation, displaying massive apoptosis of the primitive
ectoderm and resulting in early embryonic lethality (Lee
et al., 1998). In worms, rha-1 null animals are defective
in germline transcriptional control and display reduced
germ cell mitosis and defects in meiosis (Walstrom et al.,
In addition to the siRNA and Ago2 in human RISC,
a number of other proteins such as Dicer, TRBP, PACT,
and MOV10 clearly modify and enhance RISC function in
gene silencing (Rana, 2007). To identify RISC-associated
proteins, we used an approach based on affinity purifica-
tion of the guide siRNA strand incorporated into RISC in
human cells. Here we report the identification of RHA
that specifically interacts with active RISC. Depleting
RHA in cells before programming with siRNA or short hair-
pin RNA (shRNA) reduced gene silencing due to de-
creased RISC formation. RHA depletion also reduced
the recruitment of siRNA to intracellular Ago2, supporting
a role for RHA in RISC loading.
RHA Interacts with Active RISC
To identify proteins that are components of active RISC
containing the guide strand of siRNA, we developed an
affinity-purification strategy (summarized in Figure 1A).
Previously described affinity-purification strategies have
been based on coimmunoprecipitating unidentified pro-
teins with affinity-tagged RISC protein components. Our
approach used 30-LC biotinylated siRNA to isolate RISC.
To verify that this approach didn’t compromise the func-
tional integrity of RISC, we tested the ability of purified
RISC to specifically cleave radiolabeled target RNA
in vitro. Using this scheme, we purified RISC from cells
transfected with siRNA duplexes containing 30-LC biotin
on the antisense or guide strand and found that RISC spe-
cifically andpotently cleaved target RNA(Figure 1A).RISC
purified in parallel from cells programmed with siRNAs of
identical sequence but lacking 30-LC biotin on the anti-
sense strand did not cleave its target RNA. Thus, active
RISC was associated with streptavidin-coated magnetic
beads, and RISC associated with the magnetic beads
was specificallyisolated bythebiotin-streptavidin interac-
tion rather than by nonspecific interactions.
Toanalyze other components ofthe RISC, themagnetic
beads were treated with RNase. The resultant eluate con-
tained several proteins specific to extracts from cells pro-
grammed with biotinylated siRNA (Figure 1B). Mass spec-
trometric analysis of protein components eluted from the
isolated RISC-identified RHA as two bands with approxi-
mate molecular masses of ?145 and ?125 kDa. Consis-
tent with our findings, full-length RHA has a predicted
and observed molecular mass of ?142 kDa. It is currently
unclear whether the smaller RHA species we recovered
reflects posttranslational modification of the protein or
its proteolytic cleavage during preparation and purifica-
tion of the complexes. Our mass spectrometric analysis
also identified HSP90b, a protein previously identified as
an Argonaute-associated protein (Liu et al., 2004, 2005;
Maniataki and Mourelatos, 2005).
To confirm our identification of RHA as a component of
active RISC, we used an alternative approach in which
293T cells were cotransfected with biotinylated siRNA
and plasmids encoding HA-tagged RHA and myc-tagged
Ago2. Immunoblot analysis of streptavidin pullouts from
cell extracts showed that RHA was present with the guide
strand (Figure 2A). In addition, Ago2 and Dicer were pres-
ent in the RISC pulled out with the biotin-containing guide
strand. Taken together, these results show that Ago2,
Dicer, and RHA are components of active human RISC
programmed with siRNA.
To determine if siRNA was associated with RHA in hu-
man cells, we transfected 293T cells with an HA-tagged
RHA vector and immunoprecipitated the protein com-
plexes. RNA was extracted from the precipitated com-
plexes, and siRNA was detected by streptavidin-alkaline
phosphatase chemiluminescence (see Figure S1 in the
Supplemental Data available with this article online). Anti-
sense strand 30-LC biotin-conjugated siRNA was immu-
noprecipitated only from cells transfected with HA-RHA
and not with HA alone. Furthermore, in extracts of cells
transfected with HA-RHA and with nonbiotinylated siRNA,
no streptavidin binding RNA species were detected.
These results are consistent with our other findings
(Figure 2A) that RHA is a RISC-associated protein.
To determine whether or not RHA interacts with the
RISC components Dicer, TRBP, and Ago2 in the absence
ofbiotinylated siRNA,wetransfected293T cellswithFlag-
Dicer, Flag-TRBP, and Flag-Ago2. As a specificity control,
293T cells were transfected with Flag-Hexim1. Hexim1 is
an RNA binding protein that interacts with a double-
stranded region of 7SK small nuclear RNA (snRNA) and
with cyclin T1 in the inactive P-TEFb ribonucleoprotein
complex (reviewed in Zhou and Yik ). From these
transfected cells, we prepared total cell extracts (TCE)
and immunoprecipitated Flag-tagged RISC components.
Immunopurified complexes were assayed for the pres-
ence of RHA using rabbit antibodies against endogenous
RHA (Figure 2B). Immunopurified complexes from cells
transfected with Flag-Hexim1 did not contain detectable
RHA, whereas RHA was coimmunoprecipitated with
Flag-Dicer, -TRBP, and -Ago2. To verify that the observed
interactions of RHA with Dicer and Ago2 were not due to
overexpression of RHA, Dicer, or Ago2, we immunopre-
cipitated endogenous RHA from nontransfected cells.
524 Molecular Cell 26, 523–537, May 25, 2007 ª2007 Elsevier Inc.
RNA Helicase A in RISC
Figure 1. RNA Helicase A Is a RISC-Associated Protein
(A) Overview of the strategy used to purify biotinylated siGFP-RISC. RISC activity bound to streptavidin beads was assayed in vitro using a 124 nt
32P-cap-labeled synthetic RNA target. Site-specific endonucleolytic cleavage produced a 55 nt32P-cap-labeled product. Lane 1 contains strepta-
from HeLa cells transfected with GFP siRNA modified at the 30end of the antisense (guide) strand with LC-biotin.
(B) RHA and HSP90b are associated with RISC. Streptavidin beads (B) were extensively washed and treated with RNase to release RISC-associated
proteins. Supernatants (S) were resolved on 8% denaturing polyacrylamide gel and silver stained. Markers indicate molecular mass positions.
Proteins were excised from gels, eluted, and identified by mass spectrometry (MALDI QIT MS/MS).
Molecular Cell 26, 523–537, May 25, 2007 ª2007 Elsevier Inc. 525
RNA Helicase A in RISC
Supplemental Data include supplemental text and five figures and can
be found with this article online at http://www.molecule.org/cgi/
We thank members of the Rana laboratory for helpful discussions and
support and Chia-ying Chu for her assistance in illustrations. We thank
Gregory Hannon, Marc Montminy, Toshihiro Nakajima, Ramin Shie-
khattar, and Thomas Tuschl for kindly providing reagents and Dr.
John Leszyk at the University of Massachusetts Medical School Pro-
teomic Mass Spectrometry Facility for identification of proteins.
G.B.R. is the recipient of a postdoctoral fellowship award from the On-
tario HIV Treatment Network. This work was supported by grants from
the National Institutes of Health (T.M.R).
Received: September 8, 2006
Revised: February 16, 2007
Accepted: April 23, 2007
Published: May 24, 2007
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