Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila.
ABSTRACT Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.
- [show abstract] [hide abstract]
ABSTRACT: To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.Science 12/2004; 306(5700):1377-80. · 31.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Dicer is a multi-domain RNase III-related endonuclease responsible for processing double-stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into approximately 22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg(2+) or performing the reaction at 4 degrees C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4 degrees C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.The EMBO Journal 12/2002; 21(21):5875-85. · 9.82 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.Cell 08/2004; 118(1):57-68. · 31.96 Impact Factor
Current Biology 16, 530–535, March 7, 2006 ª2006 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2006.01.061
Short Interfering RNA Strand Selection
Is Independent of dsRNA Processing
Polarity during RNAi in Drosophila
Jonathan B. Preall,1Zhengying He,1Jeffrey M. Gorra,1
and Erik J. Sontheimer1,*
1Department of Biochemistry, Molecular Biology, and
2205 Tech Drive
Evanston, Illinois 60208
Short interfering RNAs (siRNAs) guide mRNA cleav-
age during RNA interference (RNAi) [1–3]. Only one
siRNA strand assembles into the RNA-induced silenc-
ing complex (RISC) , with preference given to the
strand whose 50terminus has lower base-pairing
stability [5, 6]. In Drosophila, Dcr-2/R2D2 processes
siRNAs from longer double-stranded RNAs (dsRNAs)
[7, 8] and also nucleates RISC assembly [7, 9, 10], sug-
gesting that nascent siRNAs could remain bound to
Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing
asymmetry of synthetic siRNAs and dictates strand
selection by asymmetric binding to the duplex ends
. During dsRNA processing, Dicer (Dcr) liberates
siRNAs from dsRNA ends [1, 12] in a manner dictated
cause Dcr-2/R2D2 is unlikely to sense base-pairing
asymmetry of an siRNA that is embedded within a pre-
follow the thermodynamic asymmetry rules [5, 6] or
whether processing polarity can affect strand selec-
tion . We use a Drosophila in vitro system in which
defined siRNAs with known asymmetry can be gener-
ated from longer dsRNA precursors. These dsRNAs
permit processing specifically from either the 50or
the 30end of the thermodynamically favored strand
of the incipient siRNA. Combined dsRNA-processing/
mRNA-cleavage assays indicate that siRNA strand se-
lection is independent of dsRNA processing polarity
during Drosophila RISC assembly in vitro.
Results and Discussion
Independent Variation of siRNA Base-Pairing
Asymmetry and Dcr Processing Polarity In Vitro
If the Dcr-2/R2D2 heterodimer establishes asymmetric
ing  before it can sense the relative base-pairing
cessing could modulate strand selection when siRNAs
are generated from longer precursors . To test this
possibility, we sought a biochemical system in which
the relevant variables (the polarity of Dcr processing
and the thermodynamic asymmetry of the processed
siRNAs) could be independently controlled. Tuschl and
coworkers previously showed that RNA duplexes
as short as 39 nt (but not 29 nt) are efficiently diced in
Drosophila embryo lysates and that a w20 nt 30over-
hang blocks Dcr processing at the extended end .
On the basis of these observations, we designed two
cleavage from opposite ends to generate a single, com-
mon 21 nt siRNA (see the Supplemental Experimental
Procedures in the Supplemental Data online for specific
sequences). One of these pairs forces unidirectional
processing to generate a common asymmetric siRNA
derived from the human Cyclophilin B (cyc) gene, and
the other pair generates a common symmetric siRNA
derived from the human Cu, Zn-superoxide dismutase
(sod) gene  (Figure 1A). The triggers that force Dcr ini-
tiation from the 2 nt 30-overhanging end of the sense or
antisense strand are referred to as ‘‘Dcr-R’’ or ‘‘Dcr-L,’’
respectively (Figure 1A).
more weakly base-paired end of the cyc siRNA (blue in
Figure 1A, top), and Dcr-L forces Dcr to enter from the
morestable end(redin Figure 1A,top). Inthecaseofcyc
Dcr-R, selection of the siRNA strand with the exposed 30
end is disfavored on the basis of thermodynamic asym-
metry[5,6].Elbashir etal.previously reportedprelim-
inary evidence that the processed siRNA strand with
the 30terminus exposed in the precursor is preferentially
selected during RISC assembly. Therefore, the cyc
Dcr-R dsRNA places the two reported strand-selection
parameters in conflict (Figure 1B). We included the sod
triggers to test whether dsRNA processing could impart
asymmetric strand selection on a processed siRNA that
is thermodynamically symmetric . In each case, the
sequence immediately outside of the predicted dsRNA
processing site was designed to preserve the relative
thermodynamic end stabilities in the event of Dcr-2
To confirm that the Dcr-R and Dcr-L dsRNAs gave
rise to the expected siRNA products, we 50-radiolabeled
the longer (64 nt) strand, allowing us to track the prog-
ress of the dsRNA processing step by electrophoresis
in a 15% denaturing polyacrylamide gel. All of the
dsRNAs were efficiently and specifically processed into
the predicted 21 and 22 nt siRNAs in vitro (Figure 2). We
confirmed that the shorter strand of the dsRNA was also
accurately diced by labeling this strand and observing
the appearance of the expected dsRNA processing
products (Figure S1).
Guide Strand Selection Is Independent
of Dicer Processing
strand selection, we performed a combined dsRNA-
processing/target-cleavage assay. To examine target
mRNA cleavage, we added 50-cap-radiolabeled sense
or antisense mRNA target (to assess relative siRNA
strand selection) to the dsRNA processing reactions
shown in Figure 2B and Figure S2. The targets were
added after dsRNA processing and RISC assembly had
proceeded for 15 min (for the sod triggers) or 30 min
alyzed the reactions in a 6% denaturing polyacrylamide
centration of dsRNA in each reaction was below satura-
tion for target cleavage , we normalized the fraction
of target cleaved to the extent of Dcr processing in
each reaction as determined by the fraction of 21–22 nt
and Figure S2).
The cyc and sod siRNAs directed cleavage of sense
and antisense targets with the expected levels of asym-
metry  (Figures 3A and 3C, left panels, and Figures 3B
and 3D, black circles). cyc siRNA cleaved w40% of cyc
sense target but only w5% of antisense target in 5 hr,
whereas sod siRNA cleaved w60% of both sense and
antisense targets in 2 hr. In all cases, the sod and cyc
Dcr-R triggers (Figures 3B and 3D, orange triangles) and
Dcr-L triggers (green squares) were equally efficient at
cleaving their corresponding targets and generally fol-
Unexpectedly, both of the sod 30-extended dsRNAs
directed less target cleavage than the synthetic siRNA
(Figure 3D, upper panel). Although we do not know the
reason for the reduced potency of the processed
dsRNAs with this target, it cannot be explained as a re-
sult of differential strand selection induced by the polar-
ity of Dcr processing, given that the reduction affected
both pairs of Dcr-R/Dcr-L triggers showed identical
potency in guiding cleavage of their respective sense
and antisense targets, we conclude that the direction
of Dcr processing is not likely to influence selection of
the guide strand in Drosophila embryo lysates.
To directly test whether a processed siRNA proceeds
intoRISC without dissociating from the dsRNA process-
ing machinery, we performed a chase experiment with
sod Dcr-L dsRNA that was prebound to Dcr in vitro
(Figure 4A). We first confirmed that Drosophila Dcr-2 is
capable of binding but not cleaving dsRNA when incu-
bated at 4ºC in embryo lysates (Figure S3), as is the
case for human Dcr (hDcr) . To chase newly diced
siRNA into RISC, we first preincubated sod Dcr-L
dsRNA in embryo lysate for 20 min on ice at a concen-
tration close to saturation for RISC assembly (50 nM)
. An unrelated competitor siRNA was then added
to the reaction mix on ice at various saturating concen-
trations, up to 250-fold over that of sod Dcr-L dsRNA.
The reactions were shifted to 25ºC, and radiolabeled
Figure 1. Double-Stranded RNAi Triggers that Independently Modulate Dcr Processing Polarity and siRNA Thermodynamic Asymmetry
(A) dsRNA precursors that generate asymmetric cyc siRNAs and symmetric sod siRNAs are depicted. The predicted 21 nt siRNA products are
shown above each pair of precursors. For each duplex, the sense strand (top) has its 50end on the left and its 30end on the right. Each dsRNA
comprises a 36–38 bp core duplex with a 2 nt 30overhang (which can engage Dcr ) at one end and a 26–28 nt 30overhang (which cannot
engage Dcr ) at the other end. Arrows denote sites of Dcr entry. The highly asymmetric cyc siRNA is shown with the more stably base-paired
end in red and the less stably base-paired end in blue. The symmetric sod siRNA  is shown with both ends in blue. The predicted melting free
energies of the four terminal base pairs of each siRNA [based on the Sfold Server algorithm (http://sfold.wadsworth.org/sirna.pl)] are indicated.
The regions shown in gray have no complementarity to the mRNA targets used in this study and therefore cannot make any contribution to RISC
(B) For the cyc Dcr-R dsRNA, the siRNA end that is exposed in the precursor (in blue) is less stably base-paired than the cleaved end (in red). For
this substrate, thermodynamicasymmetry is predictedto favor assembly of the antisense (bottom)strand into RISC[5, 6],whereas Dcr process-
clarity because its dsRNA substrate interactions have not been characterized.
dsRNA Processing and siRNA Strand Selection
sod sense and antisense mRNA targets were added
for 1 hr to measure RISC assembly. If the newly pro-
being released, then the unrelated siRNA would fail to
compete during RISC assembly. Regardless of whether
the competitor siRNA was added during or after the
preincubation with sod Dcr-L, we observed strong and
comparable inhibition of both sense and antisense sod
target cleavage, consistent with the idea that a freshly
diced siRNA is released from Dcr before it can initiate
RISC assembly in Drosophila embryo lysate (Figures 4B
Synthetic siRNAs Are More Potent than Dicer-
Substrate dsRNAs in Drosophila Eggs
In mammalian cells, Dcr substrates (such as short hair-
pin RNAs or 27 nt duplexes) exhibit greater silencing po-
tency than 21 nt siRNAs [16–18], suggesting that Dcr
processing might facilitate entry into the RISC assembly
pathway. To test whether the same is true in the Dro-
sophila RNAi system, we injected 0–1 hr fertilized eggs
with siRNA or 36 bp Dcr-substrate dsRNA (with a 2 nt
30overhang) directed against bicoid (bcd) mRNA. The
36 bp dsRNA was designed such that Dicer would pro-
duce an siRNA identical to the synthetic bcd siRNA
when processing was initiated from the 30end of the
guide strand. We confirmed that this 36 bp dsRNA (like
the 36 bp sod dsRNAs used in Figure 2C) was a compe-
tent dsRNA processing substrate in vitro (data not
shown). At every concentration tested, the Dcr-
substrate dsRNA was approximately half as potent at
reducing bcd mRNA levels as the corresponding ‘‘pre-
diced’’ siRNA, as measured by RT-PCR analysis (Fig-
ures 4D and 4E). The reduced activity of Dcr-substrate
dsRNAs in vivo suggests that RISC assembly and Dicer
processing are not coupled in Drosophila eggs, contrary
to findings in mammalian cells [16–18].
Implications for siRNA Strand Selection during RNAi
In Drosophila, Dcr enzymes are required for RISC as-
sembly as well as dsRNA processing [8, 9], suggesting
that the two phases of RNAi might be functionally cou-
pled in a manner that affects siRNA strand selection [1,
14]. However, our experiments indicate that Drosophila
RISC assembly and siRNA strand selection are not sig-
nificantly influenced by the dsRNA processing step
and that the thermodynamic asymmetry rules [5, 6] ap-
ply equally well with processed and unprocessed
siRNAs in this system. This suggests that Drosophila
Dcr enzymes do not channel newly generated siRNAs
directly into RISC, but rather release the siRNAs into so-
lution (or onto another factor) before they enter the RISC
Several observations have suggested that thermody-
namic asymmetry governs strand selection for pro-
cessed RNAi triggers. MicroRNAs (miRNAs) are diced
from stem-loop precursors, and in most instances only
one strand of the processed miRNA duplex is stably in-
corporated into RISC . The mature strand can be
present at either the 50or the 30end of the stem-loop,
but either way, the selected strand is generally compat-
ible with the thermodynamic asymmetry guidelines .
In addition, artificial dsRNAs introduced into plant cells
give rise to a stable set of siRNAs  that adhere to
the asymmetry rules . Similar results have been re-
ported with natural dsRNAs in plants . However, in-
terpretation of these results is difficult because the Dcr
processing polarities were not defined, and it is also
surrogate measure of RISC assembly. Furthermore,
plant cells (unlike insect and mammalian cells) export
Figure 2. Processing of Dcr-R and Dcr-L
dsRNAs in Drosophila Embryo Lysate
(A) The longer strand of each dsRNA precur-
sor (see Figure 1A) was 50-radiolabeled, an-
nealed to the unlabeled shorter strand, and
subjected to dsRNA processing. siRNAs of
21 and 22 nt accumulated as a function of
time, and the relative amounts of 21 and
22 nt products varied for each precursor.
(B) The data from (A) was quantitated with
a phosphorimager and plotted as the fraction
of precursor (defined as all RNAs greater
than w40 nt, to account for nonspecific nu-
clease degradation of the single-stranded
overhang) converted to 21–22 nt siRNAs as
a function of time.
(C) As in (A), but with the sod dsRNAs.
(D) The data from (C) were quantitated with
a phosphorimager, as in (B).
siRNAs into the vasculature to enable systemic RNAi,
and therefore the plant dsRNA processing machinery
may have specifically evolved the propensity to release
newly processed siRNAs. Thus the applicability of the
plant analyses to insects and mammals has not been
While this work was in progress, Rose et al.  char-
acterized modified w27 nt duplexes that force a defined
RNAs revealed that hDcr processing polarity can in fact
influence siRNA strand selection in transfected human
cells, although it does not completely supercede ther-
modynamic asymmetry . The reasons for the dis-
crepancy between our results and those of Rose et al.
 are not clear, although one possibility is that differ-
ent Dcr enzymes may vary in their tendencies to remain
associated with newly generated siRNAs. It is notewor-
thy that Drosophila Dcr-2 (which is primarily devoted to
the siRNA pathway [7, 8]) appears to lack the canonical
PAZ domain that normally provides Dcr with a binding
pocket for 2 nt 30overhangs [22, 23]. A PAZ domain is
present in hDcr, and mutational analyses indicate that
the hDcr PAZ domain assists with dsRNA binding and
processing when a 2 nt 30overhang is present .
The apparent lack of a PAZ domain in Dcr-2 may com-
promise its ability to remain bound to newly cleaved
siRNA. It is curious that Drosophila Dcrs are required
for RISC assembly  but do not appear to couple
dsRNA processing to siRNA strand selection, whereas
mammalian Dcrs are not required for RISC assembly
[4, 24, 25] but apparently do couple dsRNA processing
to siRNA strand selection [16–18, 26].
Finally, it remains to be determined whether Dcr en-
zymes associate with long dsRNA processing sub-
strates and siRNA RISC-assembly substrates in the
same way. This issue is undoubtedly important for un-
derstanding the functional relationship between Dcr’s
roles in the initiator and effector phases of RNAi. Crystal
structures of E. coli RNase III [27, 28], an ancestor of eu-
karyotic Dcrs, are likely to be informative. The structural
data, and models derived from them, depict a protein
that can engage dsRNAs in a dynamic fashion. A single
dsRBD on each subunit of the RNase III homodimer is
tethered to the endonuclease domain by a flexible linker
Thus, there are likely to be at least two binding modes
for dsRNA in complex with an RNase III enzyme: one in
which the dsRBD braces the RNA helix from either
side as it is channeled into the catalytic cleft [13, 29],
and another where the dsRBD holds the dsRNA above
and orthogonal to the active site . It is possible that
Dcr enzymes also exhibit alternate dsRNA binding
modes depending on whether they areactively process-
ing dsRNA or channeling siRNA into RISC. Interconver-
sion between these two conformations may require at
Figure 3. siRNA Strand Selection Is Indepen-
dent of Dcr Processing Polarity in Drosophila
(A) The different cyc dsRNAs (siRNA, Dcr-L,
and Dcr-R) were used to initiate in vitro
RNAi, as indicated at the top of each set of
lanes. The ‘‘-siRNA’’ sample was a 5 hr incu-
bation in the absence of any RNAi trigger.
50-radiolabeled mRNA and cleavage product
are indicated to the right. The reactions with
the sense (SNS) target reflect RISC activity
favored cyc antisense siRNA strand, whereas
the reactions with the antisense (ASNS)
target reflect RISC activity programmed by
the thermodynamically disfavored cyc sense
(B) The data from (A) were quantitated with a
the fraction of target cleaved vs. time. Note
that the plots for the sense and antisense tar-
gets utilize different scales on the y axes, in-
dicating w10-fold more efficient cleavage of
the sense strand in all cases.
(C) As in (A), except that the thermodynami-
cally symmetric sod triggers  were used
with their corresponding targets, and the
mRNA cleavage reactions were incubated
for 2 hr.
(D) The data from (C) was quantitated with
a phosphorimager, normalized to the effi-
ciency of dsRNA processing (as determined
from the data in Figure 2), and plotted as
the fraction of target cleaved versus time.
The plots for the sense and antisense targets
utilized the same scales on their y axes, indi-
cating comparable cleavage of the two
mRNA targets (i.e., functional symmetry).
dsRNA Processing and siRNA Strand Selection
least transient release of the siRNA product. Additional
dynamic dsRNA/protein interactions during dsRNA pro-
cessing and RISC assembly presumably involve the
dsRNA binding proteins Loquacious/R3D1 [30–32],
R2D2 , and TRBP [33, 34], which associate with
Dcr-1, Dcr-2, and hDcr, respectively. Further functional
analysis of Dcr’s PAZ, RNase III, and dsRNA binding do-
mains, aided by recent advances in the structural biol-
ogy of Dcr , will be necessary to understand Dcr’s
roles in the transition between the initiation and effector
phases of RNAi.
Supplemental Data include Supplemental Experimental Procedures
and four figures and are available with this article online at: http://
We thank Rich Carthew, John Pham, Janice Pellino, and Kevin Kim
for advice and discussions, David Levine for help with DNA con-
structs, and John Pham for comments on the manuscript. We
are grateful to Anastasia Khvorova for suggesting the use of the
asymmetric cyc siRNA and for providing us with its sequence.
This work was supported by a Cellular and Molecular Basis of Dis-
ease Training Grant to J.B.P. and National Institutes of Health
grant RO1GM072830 to E.J.S.
Received: August 19, 2005
Revised: January 17, 2006
Accepted: January 20, 2006
Published: March 6, 2006
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