P68 RNA helicase was identified as being required for unwinding the human let-7 microRNA precursor duplex. Recombinant P68 RNA helicase was shown to unwind the let-7 duplex in vitro. Knockdown of P68 inhibited let-7 microRNA function, indicating P68 is essential for loading let-7 into the silencing complex. This study identifies P68 RNA helicase as playing a key role in the human let-7 microRNA pathway.

1. Shubert-Coleman and Henry Furneaux
David W. Salzman, Jonathan
Gene Expression
Required for let-7-directed Silencing of
let-7 MicroRNA Precursor Duplex and Is
P68 RNA Helicase Unwinds the Human
RNAs:
RNA-Mediated Regulation and Noncoding
doi: 10.1074/jbc.M705054200 originally published online August 27, 2007
2007, 282:32773-32779.J. Biol. Chem.
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2. P68 RNA Helicase Unwinds the Human let-7 MicroRNA
Precursor Duplex and Is Required for let-7-directed
Silencing of Gene Expression*
Received for publication,June 20, 2007, and in revised form, July 31, 2007 Published, JBC Papers in Press,August 27, 2007, DOI 10.1074/jbc.M705054200
David W. Salzman, Jonathan Shubert-Coleman, and Henry Furneaux1
From the Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center,
Farmington, Connecticut 06030
MicroRNAs are short, single-stranded RNAs that arise from a
transient precursor duplex. We have identified a novel activity
in HeLa cell extracts that can unwind the let-7 microRNA
duplex. Using partially purified material, we have shown that
microRNA helicase activity requires ATP and has a native
molecular mass of ϳ68 kDa. Affinity purification of the unwind-
ing activity revealed co-purification of P68 RNA helicase.
Importantly, recombinant P68 RNA helicase was sufficient to
unwind the let-7 duplex. Moreover, like its native homolog, P68
RNA helicase did not unwind an analogous small interfering
RNA duplex. We further showed that knockdown of P68 inhib-
ited let-7 microRNA function. From our data, we conclude that
P68 RNA helicase is an essential component of the let-7
microRNA pathway, and in conjunction with other factors, it
may play a role in the loading of let-7 microRNA into the silenc-
ing complex.
It is now recognized that microRNAs play a critical role in the
regulation of gene expression and that their deregulation may
underlie many human diseases (1–5). Thus, it is important to
understand how they are incorporated into the protein cofactor
complexes that are essential for their suppressive activity.
MicroRNAs are initially transcribed as precursor RNAs, which
fold into hairpin structures (6). The acknowledged first step of
microRNA maturation is the specific endonucleolytic cleavage
of the pri-microRNA near the base of the stem loop, by Drosha
(7). The Drosha cleavage product is typically a 70-nucleotide
stem loop RNA, containing a two-nucleotide overhang at the 3Ј
end and a recessed 5Ј-phosphate (8–10). This product is a sub-
strate for a second enzyme called Dicer (11). Dicer cleaves both
strands of the hairpin precursor ϳ21 nucleotides from the end
of the stem loop creating a 19-nucleotide paired duplex with
two-nucleotide overhangs at the 3Ј ends: the transient
microRNA duplex (12–14).
It is now clear that the silencing activities of microRNAs are
effected by a novel class of RNA-binding proteins called the
Argonaute family (15–18). However, the affinity of these pro-
teins for duplex RNA is much less than that observed for single-
stranded RNA (15, 19). Moreover, analysis of Argonaute/RNA
complexes in the cell typically reveals the association of only
one strand (the guide strand) of this transient duplex (20). Thus,
there are likely to be additional factors, which unwind the tran-
sient duplex and confer specificity in uptake into Argonaute.
Studies on structurally different, but conceptually analogous,
siRNA2
duplexes have revealed that this specificity in uptake
may be conferred by a heterodimer of Dicer and R2D2 in which
the stable end of the duplex is bound by R2D2 and Dicer facil-
itates the loading of the loosely paired 5Ј end of the guide strand
into Argonaute (21–24). Although the unwinding is likely facil-
itated by the cleavage of the passenger strand by Argonaute (25,
26), RNA helicase A has also recently been implicated in load-
ing of the guide strand of siRNA (27). Much less is known about
the factors that are likely required to load the guide strand of the
analogous microRNA duplex into Argonaute complexes. In
contrast to siRNA duplexes, microRNA duplexes typically con-
tain unpaired bulges, which likely preclude the cleavage of the
passenger strand by Argonaute (26).
An affinity-purified complex that contains Argonaute2 and
Dicer has been shown to direct the cleavage of a target mRNA
directed by a let-7 hairpin precursor (19, 28). Although this
argues that Dicer might be necessary for the unwinding of the
microRNA transient duplex, microRNA duplexes can still
silence expression in cells that lack Dicer (29). In any event,
neither the siRNA-directed nor the microRNA duplex-directed
cleavage of a mRNA has been successfully reconstituted with
recombinant proteins, and thus, it is likely that additional fac-
tors are required for this step.
In the present study, we have identified an activity from
human cells that promotes the ATP-dependent unwinding of
the human let-7 microRNA precursor duplex. Further charac-
terization suggested that this activity corresponded to the pre-
viously described P68 RNA helicase (30–32). Indeed, we found
that recombinant P68 RNA helicase was sufficient to unwind
the let-7 microRNA precursor duplex. Importantly, a transient
knockdown of P68 abrogated let-7-directed suppression of
gene expression and indicates that P68 RNA helicase is indeed
required to facilitate the uptake of duplex let-7 microRNA into
the silencing complex.
* This work was supported by National Institutes of Health Grants R03
DA022226 and P01 HL70694. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must there-
fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-
tion 1734 solely to indicate this fact.
1
To whom correspondence should be addressed: University of Connecticut
Health Center, 236 Farmington Ave., Farmington, CT 06030. Tel.: 860-679-
2374; Fax: 860-679-1862; E-mail: furneaux@nso.uchc.edu.
2
The abbreviations used are: siRNA, small interfering RNA; GAPD, glyceralde-
hyde-3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogen-
ase; AMP-PNP, adenosine 5Ј-(␤,␥-imino)triphosphate.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 45, pp. 32773–32779, November 9, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
NOVEMBER 9, 2007•VOLUME 282•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32773at YALE UNIV | Kline Science Library on August 21, 2013http://www.jbc.org/Downloaded from

3. EXPERIMENTAL PROCEDURES
HeLa S3 cells were obtained from the National Cell Culture
Center (Minneapolis, MN). Synthetic RNAs and siRNAs were
obtained from Dharmacon Research Inc. (Lafayette, CO).
Luciferase reporter plasmids were provided by the David Bartel
laboratory. The His-P68 plasmid was provided by the Zhi-Ren
Liu laboratory. Anti-P68 monoclonal antibody (PAB204) was
obtained from Upstate Biochemicals, and monoclonal antibod-
ies against GAPD and Vimentin were obtained from Abcam.
Preparation of HeLa Cell Extract—HeLa S3 cells (National
Cell Culture Center) were resuspended in hypotonic buffer (50
mM Tris, pH 7.5, 10 mM KCl, 5 mM dithiothreitol). The swollen
cells were homogenized, and KCl, MgCl2, and glycerol were
added to final concentrations of 100 mM, 2 mM, and 10%,
respectively. The homogenate was centrifuged at 500 ϫ g for 10
min. The supernatant was removed,
and the pellet was resuspended in
buffer A (50 mM Tris, pH 7.5, 2 mM
MgCl2, 5 mM dithiothreitol, 10%
glycerol). KCl was added dropwise
to a final concentration of 400 mM.
The homogenate was centrifuged at
10,000 ϫ g for 10 min. The resultant
nuclear extract was stored at Ϫ80 °C
in aliquots.
RNA Affinity Chromatography—
0.5 ml of avidin A beads (Vector
Laboratories) were incubated with
36 ␮mol of biotinylated let-7 pre-
cursor hairpin RNA in 0.6 ml vol-
ume with buffer A (50 mM Tris, pH
7.5, 0.01% Nonidet P-40, 10% glyc-
erol) at 4 °C for 8 h. Beads were
washed with buffer A containing 1 M
NaCl and then equilibrated with
buffer A containing 50 mM NaCl.
Nuclear extract was applied to the
column and washed with buffer A
containing 50 mM NaCl. The col-
umn was then eluted with buffer A
containing a 50 mM stepwise 0.05
M-0.8 M NaCl gradient.
Preparation of Recombinant His-
P68 RNA Helicase—Recombinant
His-P68 was prepared as described
previously (31). In short, His-P68
was induced in BL21 (DE3) cells
with 0.1 mM isopropyl-1-thio-␤-D-
galactopyranoside at 37 °C for 6 h.
Bacterial pellets were resuspended
in lysis buffer (50 mM Tris, pH 8.0,
0.1 M NaCl, 0.5 mM EDTA, pH 8.0)
and were lysed by the addition of
lysozyme (0.2 mg/ml) and Triton
X-100 (1%). The resulting superna-
tant was applied to a nickel-nitrilo-
triacetic acid agarose column. The
column was first washed with lysis
buffer containing 20 mM imidazole followed by lysis buffer con-
taining 20 mM imidazole and 0.15 mM NaCl. Recombinant His-
P68 was then eluted with buffer containing 250 mM NaCl and
250 mM imidazole.
Preparation of Labeled MicroRNA Precursor Duplex—The
let-7 guide strand was labeled using T4 polynucleotide kinase
and [␥-32
P]ATP (Amersham Biosciences). After phenol-chlo-
roform extraction, it was annealed (65 °C for 5 min and then
37 °C for 25 min) to a 5-fold excess of let-7 passenger strand.
The duplex was then gel-purified and stored in 50 mM Tris, pH
7.5, and 0.2 M potassium acetate.
Precursor Duplex-unwinding Assay—Reaction mixtures
(0.02 ml) contained labeled let-7 precursor duplex (1 nM), 50
mM Tris, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 5 mM ATP, and
nuclear extract or recombinant P68 RNA helicase as indicated.
FIGURE 1. Identification of a novel activity from human cells that unwinds the let-7 duplex. A, sequence
and structure of the Let-7 microRNA duplex substrate. The microRNA or guide strand is the bottom strand.
B,theduplexlet-7substrateanditssinglestrandedderivative(producedbyheatpenetration)asanalyzedbynative
gel electrophoresis. C, 32
P-labeled let-7 duplex was incubated with increasing amounts of crude extract made from
HeLacells(amountsindicated).Reactionsweredeproteinizedandanalyzedforthepresenceofsingle-strandedRNA
using native polyacrylamide gel electrophoresis. The asterisk indicates the addition of heat-inactivated extract.
D, crude HeLa cell extract was fractionated using a Sephadex S-200 column. Fractions (2 ␮l) were assayed for
unwinding activity, using 32
P-labeled let-7 duplex. Molecular mass markers are: alcohol dehydrogenase (150 kDa),
bovineserumalbumin(68kDa),andcytochromec(12.4kDa).E,32
P-labeledlet-7duplexwasassayedforunwinding
activity,usingfraction20fromtheSephadexS-200column(2␮g)intheabsenceorpresenceofdifferentnucleoside
cofactors (as indicated). The asterisk indicates a reaction containing a heat-inactivated Sephadex fraction.
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4. After 30 min at 37 °C, the reaction was terminated by the addi-
tion of SDS-EDTA buffer (50% glycerol, 0.1 M Tris, pH 8.0, 0.5%
SDS, 20 mM EDTA, 0.1% Nonidet P-40, 0.1% each bromphenol
blue, and xylene cyanol) and analyzed by 15% native polyacryl-
amide gel electrophoresis.
Luciferase Assay of MicroRNA Function—HeLa cells were
transfected using Lipofectamine 2000 in a 12-well plate using
the indicated amounts of firefly luciferase reporter plasmid and
either ␤-galactosidase or Renilla luciferase plasmid. Firefly and
either ␤-galactosidase or Renilla luciferase activities were
assayed 36–48 h after transfection using the Dual Luciferase
assay (Promega). Firefly activity was normalized by cotransfec-
tion of either ␤-galactosidase or Renilla plasmids to control for
transfection efficiency.
RESULTS
The Identification of an ATP-dependent Activity That Can
Unwind the let-7 MicroRNA Duplex—We elected to use the
human let-7 duplex (Fig. 1A) as a model since let-7 is absolutely
conserved between worms and humans and has been biochem-
ically studied in many systems (12, 33–39). This choice there-
fore permits the ready comparison of any identified human pro-
tein co-factors with those identified in other organisms.
However, one disadvantage of the human let-7 duplex is that a
discernible level of single strand is produced merely on incuba-
tion at 37 °C. Thus, in all experiments, we included a negative
comparison control (a heat-inactivated corresponding cellular
fraction) so that we could clearly
distinguish any cellular helicase
activity from the enzyme-indepen-
dent background. Fig. 1B shows that
the let-7 duplex was unwound on
incubation with nuclear extract
made from HeLa cells. The extent of
unwinding titrated with the amount
of extract added to the reaction. To
extend this observation and to
establish the native molecular
weight of the activity, we fraction-
ated the extract using a Sephadex
S-200 column. Fractions were col-
lected and assayed for helicase
activity. There was a minor high
molecular weight species; however,
the majority of the unwinding activ-
ity eluted in an inclusion volume
consistent with a native molecular
mass of 68 kDa (Fig. 1C).
Next, using this partially purified
material, we investigated whether
the unwinding activity required
ATP. Little unwinding was seen in
the absence of ATP, whereas the
addition of ATP markedly stimu-
lated the reaction and appeared to
saturate at 5 mM. In addition, no
unwinding activity was seen in reac-
tion mixtures containing AMP-
PNP, a non-hydrolyzable ATP analog (Fig. 1D). From these
observations,weconcludedthatwehaveidentifiedanovelATP-
dependent activity in HeLa cell extract that is capable of
unwinding the let-7 microRNA duplex and that this activity has
a native molecular mass of ϳ68 kDa.
P68 RNA Helicase Co-purifies with the MicroRNA Duplex-
unwinding Activity—Our initial observations suggested that
the activity that unwinds the let-7 duplex precursor might
correspond to P68 RNA helicase. P68 RNA helicase is an
ATP-dependent RNA-unwinding enzyme of 68 kDa that has
been found to be a subunit of the Drosha-processing com-
plex (8, 30–32, 40). Accordingly, we elected to affinity-purify
our unwinding activity and investigate whether it co-puri-
fied with P68 RNA helicase. We prepared an affinity column
by immobilizing a biotinylated hairpin sequence containing
the let-7 passenger and guide sequences (Fig. 2A) to avidin
beads.
HeLa cell extract was applied, the column was washed with
low salt, and bound protein was eluted using a step gradient
from 0.05 to 0.8 M NaCl (Fig. 2B). A significant portion of the
microRNA helicase activity was retained and eluted at 200–250
mM NaCl (Fig. 2C). Next, we assayed for the presence of P68
RNA helicase by Western blot and found that it was precisely
coincident with the unwinding activity (Fig. 2D). Thus, we con-
cluded that P68 was indeed a strong candidate for the
microRNA-unwinding activity.
FIGURE 2. Purification of the let-7 microRNA helicase activity yields P68 RNA helicase as a likely candidate.
A,thesequenceandlikelystructureofthelet-7hairpinRNAusedtopurifytheactivity.BiindicatestheBiotinmoiety.
B, HeLa cell extract was applied to a let-7 hairpin affinity column and eluted with a salt gradient. C, fractions (2 ␮l)
wereassayedforunwindingactivity,using32
P-labeledlet-7duplex.D,fractionsfromthelet-7hairpinaffinitycolumn
(2 ␮l) were analyzed via Western blot for P68 RNA helicase using PAb204. Molecular size markers are
indicated in kDa.
P68 RNA Helicase Is Required for Silencing of Gene Expression
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5. Recombinant P68 RNA Helicase Is Sufficient to Unwind the
let-7 MicroRNA Duplex—Next, we investigated whether
recombinant P68 was sufficient to unwind the let-7
microRNA duplex, and if so, whether its properties resem-
bled those displayed by the native activity. First, we investi-
gated the structural features of the microRNA duplex that
are necessary for unwinding by the native activity. We syn-
thesized two mutant derivatives of the let-7 duplex. In the
first mutant (mutant 1), nucleotide substitutions were made
so that every nucleotide of the microRNA guide strand was
annealed to the passenger. This mutant is a “bad” siRNA in
which the 5Ј end of the guide strand is annealed to the pas-
senger strand. Previous studies
have shown that the guide strand
of such an siRNA will not be
readily incorporated into a silenc-
ing complex (21, 22, 26, 41). This
mutant was a very poor substrate;
virtually no unwinding activity
was noted even on incubation with
saturating amounts of affinity-pu-
rified unwinding activity (Fig. 3, A
and B).
Next, we synthesized a mutant
that lacked the internal bulges of
the microRNA duplex yet retains
the unpaired structure of the 5Ј
end of the guide strand. This
mutant (mutant 2) is analogous to
a “good” siRNA in which the guide
strand is readily incorporated into
the silencing complex (41). Impor-
tantly, this mutant was also a very
poor substrate and indicates that
the unwinding activity recognized
the internal bulges and therefore
can distinguish between an siRNA
duplex and a microRNA duplex.
Indeed, the importance of the
internal bulges was illustrated by
the observation that a third
mutant, in which the bulges were
retained and the 5Ј end of the
guide was annealed to the passen-
ger strand, exhibited significant
unwinding Fig. 3, A and B).
Importantly, recombinant P68
RNA helicase also displayed a
marked preference for a microRNA
duplex, and the critical role of the
internal bulges was similarly evi-
dent. Thus, we concluded that P68
RNA helicase is sufficient to unwind
the let-7 duplex, and like the affini-
ty-purified activity, it prefers a
microRNA duplex to an siRNA
duplex (Fig. 3, A and C).
P68 RNA Helicase Is Required
for let-7 MicroRNA Function in HeLa Cells—If P68 RNA
helicase was required for the unwinding of the let-7
microRNA duplex, one would predict that its down-regula-
tion would prevent loading of let-7 microRNA into the
silencing complex and that significant inhibition of
microRNA activity would result. To test this hypothesis, we
utilized a reporter plasmid (pIS-Lin41(s) (42)) that contains
a previously characterized let-7-response element found in
lin-41 mRNA. Indeed, transfection of this plasmid into HeLa
cells resulted in a 22-fold repression when compared with
normalized luciferase activity in comparison with that
expressed by the parental plasmid that lacks the response
FIGURE 3. Purified recombinant His-P68 RNA helicase is sufficient to unwind the let-7 microRNA duplex.
A, 32
P-labeled let-7 duplexes were analyzed for helicase activity using affinity-purified material (left panel,
amounts indicated) or recombinant His-P68 (middle panel, amounts indicated). I, indicates the input into each
reaction. ⌬T indicates heat-denatured input. The sequence and structure of each let-7 duplex are indicated
next to the companion assay. Mutations along the passenger strand are indicated in red. * and N indicate heat
denaturedandnativeprotein,respectively.B,quantificationoftheunwindingactivityexhibitedbytheaffinity-
purified material. C, quantification of the unwinding activity exhibited by recombinant His-P68.
P68 RNA Helicase Is Required for Silencing of Gene Expression
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6. element (Fig. 4A). Evidence that this suppression was exerted
by let-7 was provided by the observation that this suppression
was alleviated by AntagomiRs against let-7 but not by
AntagomiRs against an irrelevant microRNA (Fig. 4A).
Moreover, the suppressive activ-
ity of the element was attenuated
by mutations that compromise the
annealing of let-7 (Fig. 4B). In the
following experiment, we have
used the comparison of the lucif-
erase activity between the wild
type (WT) and mutant (MUT) let-
7-response elements as a measure
of let-7 activity. siRNA-mediated
down-regulation of P68 RNA heli-
case, but not GAPDH, attenuated
the activity of let-7 microRNA
(Fig. 4B). Importantly, GAPDH
was successfully down-regulated
as shown by Western blot analysis
using Vimentin as a loading con-
trol (Fig. 4C). Thus, we conclude
that P68 RNA helicase is indeed
required for microRNA activity
and is likely required for the incor-
poration of the microRNA into the
Argonaute2 complex.
DISCUSSION
The single-stranded small RNA-
directed silencing of mRNA has
been reconstituted with recombi-
nant Argonaute2 (18). However, the
precursor microRNA duplex-di-
rected silencing of mRNA has not
yet been reconstituted with the can-
didate recombinant proteins. Thus,
it is likely that other factors remain
to be discovered. In these studies,
we have identified an ATP-depend-
ent unwinding activity that specifi-
cally unwinds the let-7 microRNA
duplex yet exhibits little activity
on a derived siRNA duplex. This
observation reinforces the current
perception that the guide strands
of siRNA and microRNA duplexes
arrive in Argonaute2 complexes
through different pathways (20,
22, 26). Indeed, it will be interest-
ing to see whether RNA helicase A,
which has been implicated in the
unwinding of the siRNA duplex
(27), is also capable of unwinding
some microRNA duplexes.
Our size fractionation analysis
and affinity purification studies sug-
gested that a principal component
of the unwinding activity corresponds to the P68 RNA helicase.
P68 was originally identified in human cells due to its coinci-
dental reactivity with a monoclonal antibody directed against
SV40 large T antigen (40).
FIGURE 4. P68 RNA helicase is required for let-7 microRNA function in HeLa cells. A, HeLa cells were
co-transfected with a ␤-galactosidase plasmid (100 ng) and either the parental plasmid (pIS-0) or a let-7-
responsive reporter (lin41(s) WT (where WT indicates wild type)) (100 ng, as indicated), along with AntagomiRs
against let-7 or Mir-16 (amounts indicated). Cells were incubated for 36 h and subsequently analyzed for
luciferase activity. Results are normalized to cotransfected plasmid encoding ␤-galactosidase. B, HeLa cells
wereco-transfectedwithaRenillaluciferasereporter(100ng)andthepIS-0,lin-41(s)WT,orlin-41(s)MUT(where
MUT indicates mutant) (42) luciferase reporter (100 ng, as indicated) along with siRNA directed against either
P68 RNA helicase or GAPDH (amounts indicated). Cells were incubated for 40 h and analyzed for luciferase
activity. Results are normalized to Renilla luciferase. C, left panel, HeLa cells treated with siRNA against P68 RNA
helicase or GAPDH (amounts indicated) were analyzed for protein via Western blot. Protein levels were nor-
malized to Vimentin.
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7. P68 was originally believed to be an RNA helicase due to its
homology to the DEAD box of eIF-4A, a well characterized
RNA helicase (43, 44). When P68 was assayed for unwinding
activity, it was found to be an ATP-dependent RNA helicase,
which can unwind RNA duplexes in both 3Ј to 5Ј and 5Ј to 3Ј
directions (30–32). The substrates unwound by P68 RNA heli-
case range in size from 22 to 175 nucleotides in length and
contain overhangs of varied lengths ranging from 6 to 185
nucleotides long (30–32). Thus, our observations are consist-
ent with the known properties of P68 RNA helicase.
P68 RNA helicase has been implicated in many cellular func-
tions. In some cases, for example, in its perceived role as a tran-
scriptional regulator, this function does not require helicase
activity (45). In most cases, however, its touted role in mRNA
splicing, rRNA processing, and mRNA decay requires the
integrity of the helicase domain (46–48). Our studies here lead
us to speculate that P68 RNA helicase might regulate the
expression of many microRNAs with a consequent pleiotropic
effect upon cellular function. It is possible that this function
might accommodate many of the previously ascribed functions
in RNA metabolism. In any event, the regulation of microRNA
activity would be consistent with its well described role in cel-
lular proliferation. However, it remains possible that P68 RNA
helicase may only unwind particular subclasses of microRNA
duplexes, and its role in a particular cell function may be pecu-
liar to the miRNAs that are expressed in a given cell type.
Our contention that P68 RNA helicase plays a role in the
unwinding of the let-7 microRNA duplex is strengthened by the
previous observation that it is a subunit of the affinity-purified
Drosha-processing complex (48). In addition, it has been
recently demonstrated that mouse embryonic fibroblasts that
lack P68 RNA helicase are compromised in their expression of
many microRNAs (48). Importantly, we show here, for the first
time, that a recombinant RNA helicase is sufficient to unwind a
microRNA precursor duplex. From this key observation, we
would propose that P68 RNA helicase might drive the selective
uptake of the let-7 guide strand into the silencing complex. So
far, however, our preliminary attempts to directly demonstrate
this using purified, recombinant proteins have not been suc-
cessful. However, the present studies have identified an essen-
tial co-factor that may ultimately facilitate the reconstitution of
this critical step.
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