scientific report
`
`scientificreport
`
`Cleavage of the siRNA passenger strand during RISC
`assembly in human cells
`Philipp J.F. Leuschner1, Stefan L. Ameres2, Stephanie Kueng3 & Javier Martinez1+
`1Institute of Molecular Biotechnology of the Austrian Academy of Sciences, IMBA, Vienna, Austria, 2Max F. Perutz Laboratories,
`Department of Biochemistry, University of Vienna, Vienna, Austria, and 3Institute of Molecular Pathology, IMP, Vienna, Austria
`
`A crucial step in the RNA interference (RNAi) pathway involves
`the assembly of RISC, the RNA-induced silencing complex. RISC
`initially recognizes a double-stranded short
`interfering RNA
`(siRNA), but only one strand is finally retained in the functional
`ribonucleoprotein complex. The non-incorporated strand, or
`‘passenger’ strand, is removed during the assembly process and
`most probably degraded thereafter. In this report, we show that
`the passenger strand is cleaved during the course of RISC
`assembly following the same rules established for the siRNA-
`guided cleavage of a target RNA. Chemical modifications
`impairing the cleavage of the passenger strand also impair the
`cleavage of a target RNA in vitro as well as the silencing of
`a reporter gene in vivo, suggesting that passenger strand removal
`is facilitated by its cleavage during RISC assembly. Interestingly,
`target RNA cleavage can be rescued if an otherwise non-
`cleavable passenger strand shows a nick at the scissile phospho-
`diester bond, which further indicates that the cleavage event
`per se is not essential.
`Keywords: passenger strand; RNAi; Argonaute; RISC
`EMBO reports (2006) 7, 314–320. doi:10.1038/sj.embor.7400637
`
`INTRODUCTION
`RNA interference (RNAi) is a post-transcriptional gene-silencing
`phenomenon that occurs in many eukaryotic organisms following
`stimulation by double-stranded RNA (dsRNA; Fire et al, 1998). In
`general, the RNAi response (reviewed by Filipowicz, 2005) can be
`divided into two distinct steps. The first step, called the assembly
`phase, comprises the recognition of a dsRNA molecule and its
`processing into B21-nucleotide (nt) RNA molecules, termed short
`interfering RNAs (siRNAs), by the RNase III-like enzyme Dicer
`(Hammond et al, 2000; Zamore et al, 2000; Bernstein et al, 2001).
`
`1Institute of Molecular Biotechnology of the Austrian Academy of Sciences, IMBA,
`Dr-Bohr-Gasse 3-5, 1030 Vienna, Austria
`2Max F. Perutz Laboratories, Department of Biochemistry, University of Vienna,
`Dr-Bohr-Gasse 9/5, 1030 Vienna, Austria
`3Institute of Molecular Pathology, IMP, Dr-Bohr-Gasse 7, 1030 Vienna, Austria
`+Corresponding author. Tel: þ 43 1 79044 4860;
`E-mail: javier.martinez@imba.oeaw.ac.at
`
`Received 3 November 2005; revised 12 December 2005; accepted 21 December
`2005; published online 20 January 2006
`
`siRNAs are then shuttled into the RNA-induced silencing complex
`(RISC), a ribonucleoprotein complex composed of an Argonaute
`protein (Elbashir et al, 2001a; Lee et al, 2004; Pham et al, 2004;
`Tomari et al, 2004a, b) and a single-stranded guide RNA. The
`selection of the RNA strand to be incorporated is governed by the
`thermodynamic profile of the siRNA duplex termini (Khvorova
`et al, 2003; Schwarz et al, 2003). In the second phase, also known
`as the effector phase, RISC uses this single-stranded RNA molecule
`as a guide to endonucleolytically cleave complementary RNAs
`(Hammond et al, 2000; Nyka¨nen et al, 2001; Elbashir et al, 2001a;
`Martinez et al, 2002). Although RISC-mediated target RNA
`cleavage is very well studied (Haley & Zamore, 2004; Liu et al,
`2004; Martinez & Tuschl, 2004; Meister et al, 2004; Schwarz et al,
`2004; Song et al, 2004), the final steps of the assembly process of
`RISC are still a matter of debate, especially how the ‘guide’ strand
`is separated from the passenger strand.
`Whereas in mammalian cells only few studies exist on the
`assembly of RISC (Pham & Sontheimer, 2005), this process has
`been extensively studied in Drosophila melanogaster (Okamura
`et al, 2004; Tomari et al, 2004a, b), in which the unwinding of
`the siRNA has been shown to depend on the presence of Ago2
`(Okamura et al, 2004; Tomari et al, 2004b). Such studies have
`so far focused on the siRNA strand that
`is incorporated into
`RISC, referred to as the ‘guide strand’. In contrast, the fate of the
`non-incorporated strand, or ‘passenger strand’, has mostly been
`neglected. In this report, we investigate the role and fate of the
`passenger strand during RISC assembly in HeLa cells both in vitro
`and in vivo, and focus on its implications on silencing. We show
`that
`for its efficient removal,
`the passenger strand has to be
`0
`cleavable at its ‘natural’ site, that is, at 10 nt from the 5
`-phosphate
`of the guide strand. If this cleavage step is blocked, the siRNA
`duplex still becomes loaded into RISC, but target RNA cleavage
`is severely impaired owing to the non-efficient removal of the
`passenger strand.
`
`RESULTS AND DISCUSSION
`Modified passenger strands impair target RNA cleavage
`It was previously shown that affinity-purified human RISC is able
`to cleave synthetic, short, non-capped RNAs (Martinez & Tuschl,
`0
`2004). Interestingly, a short target RNA containing a 2
`-O-methyl
`ribose at guanosine 9 (G9, the nucleotide immediately upstream of
`
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`&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
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`Alnylam Exh. 1078
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`RISC assembly in human cells
`P.J.F. Leuschner et al
`
`scientific report
`
`Mismatch 5–8
`
`Mismatch 9–12
`
`Mismatch 13–16
`
`Mismatch 17–19
`
`Mismatch 17–19
`
`Mismatch 13–16
`Mismatch 9–12
`Mismatch 5–8
`
`4S-U3
`
`4S-U10
`
`4S-U16
`
`4S-U20
`
`E
`
`F
`
`4S-U20
`4S-U16
`4S-U10
`4S-U3
`
`C
`
`D
`
`3′
`
`5′
`
`Unmodified
`
`G9-A10-P-thioate
`G9-2′-O-methyl
`A10-2′-O-methyl
`A11-2′-O-methyl
`
`A11-2′-O-methyl
`A10-2′-O-methyl
`G9-2′-O-methyl
`G9-A10-P-thioate
`Unmodified
`
`21-nt
`passenger
`strand
`
`A
`
`B
`
`RNA substrate
`
`5′ cleavage
`product
`
`Percentage of cleavage 6.8
`
`1.1 0.9 5.7 5.6
`
`6.0 1.3 6.4 6.9
`
`5.8 1.9 5.5 7.3
`
`Fig 1 | Chemical modifications on the passenger strand impair cleavage of a target RNA. (A) Graphical representation of short interfering RNAs
`(siRNAs) containing unmodified or chemically modified passenger strands. Passenger strands are shown in grey and guide strands in black in this
`0
`-O-methyl modifications are depicted as circles and phosphorothioates as triangles. (B) Phosphorimaging of cleavage reactions using siRNAs
`report. 2
`0
`depicted in (A) resolved on a 6% denaturing polyacrylamide gel. Arrows point to the RNA substrate and the labelled 5
`-cleavage product. The
`percentage of cleaved target RNA is indicated at the bottom of the gel. (C) Graphical representation of siRNAs containing 4S-U substitutions
`(indicated as bars) at different positions on the passenger strand. (D) Phosphorimaging of cleavage reactions using siRNAs depicted in (C).
`(E) Graphical representation of siRNAs containing 4-nt mismatches at different positions on the passenger strand. (F) Phosphorimaging of cleavage
`reactions using siRNAs depicted in (E).
`
`the cleavage site) was poorly cleaved, probably owing to steric
`hindrance caused by the bulky methyl group. A deoxyribose at G9
`0
`and 2
`-O-methyl ribose groups 1 and 2 nt downstream of the
`cleavage site were, however, well tolerated.
`We reasoned that RISC, during its assembly on the guide
`strand, might regard a passenger strand as its first RNA target, in
`a similar manner as affinity-purified RISC recognizes and cleaves
`a short target RNA. In this model, a chemically modified short
`RNA that cannot be cleaved by affinity-purified RISC should also
`not be cleaved when present as a passenger strand in an siRNA.
`We used HeLa cytoplasmic extracts and monitored the effect
`of these and other modifications on target RNA cleavage when
`present on the passenger strand of an siRNA duplex.
`We observed a greater than sixfold reduction in the cleavage
`of a complementary target RNA when the passenger strand
`contained a phosphorothioate bond between G9 and A10, a
`modification that was shown to impair cleavage of a substrate
`0
`-O-methyl ribose at G9
`RNA (Schwarz et al, 2004), or featured a 2
`0
`(Fig 1A, and Fig 1B, lanes 2,3). However, 2
`-O-methyl ribose
`groups positioned 1 or 2 nt downstream of the cleavage site (A10
`and A11) did not impair target RNA cleavage, as was the case for
`an unmodified siRNA (Fig 1A, and Fig 1B, lanes 1,4,5). A similar,
`B6-fold reduction in target RNA cleavage was obtained with a
`passenger strand, in which the adenine residue downstream of the
`predicted cleavage site (A10) was substituted by a 4-thio-uridine
`(4S-U; Fig 1C, and Fig 1D, lane 2). This modification, when
`present in a short target RNA, abolished cleavage by affinity-
`purified RISC, most probably owing to a geometrical distortion in
`
`base pairing (supplementary Fig S1 online). 4S-U residues at non-
`central positions did not severely interfere with the cleavage of the
`target RNA (Fig 1C, and Fig 1D, lanes 1,3,4). Finally, we tested
`the effect of passenger strands featuring mismatches at different
`positions on the cleavage of a guide-complementary target RNA.
`A central 4-nt mismatch reduced the cleavage efficiency by a
`factor of 3 when compared with a full complementary siRNA
`(Fig 1E, and Fig 1F, lane 2), whereas non-central mismatches
`did not significantly affect the efficiency of cleavage (Fig 1E, and
`Fig 1F,
`lanes 1,3,4). These results indicate that modifications
`at the putative cleavage site on the passenger strand severely
`0
`-O-methyl
`impair the assembly of functional RISC. Interestingly, 2
`modifications at central positions on the guide strand had no effect
`on target RNA cleavage (data not shown), underlining that the
`impairment of target RNA cleavage is due to the passenger strand
`being rendered non-cleavable.
`
`The passenger strand is cleaved during RISC assembly
`As (i) RISC properly recognizes siRNAs featuring non-cleavable
`passenger strands (supplementary Fig S2A,B online), and (ii) Ago2
`interacts with both strands of
`the duplex (supplementary Fig
`S2C,D online), we reasoned that the inhibition of target RNA
`cleavage might be explained by RISC being unable to effectively
`remove a passenger strand that cannot be cleaved, rather than
`failing to assemble on a modified siRNA. In principle, it could
`also be the case that a non-cleavable passenger strand acted as
`a suicide target on re-binding functional RISC. We can, however,
`rule out this possibility, as a non-cleavable passenger strand is not
`
`&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
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`EMBO reports VOL 7 | NO 3 | 2006
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`scientific report
`
`RISC assembly in human cells
`P.J.F. Leuschner et al
`
`Predicted
`cleavage
`
`A
`
`32P
`
`32P
`
`32P
`
`9-nt cleavage
`product
`
`3′
`
`5′
`
`Unmodified
`
`G9-2′-O-methyl
`
`G9-A10-P-thioate
`
`B
`
`32P
`
`32P
`
`C
`32P
`
`Predicted
`cleavage
`
`9-nt cleavage
`product
`
`11-nt cleavage
`product
`
`3′
`
`5′
`
`Compensated
`
`Mismatch
`
`Blunt
`
`D
`
`5′-32P-21-nt
`passenger strand
`T1
`
`Time (min)
`
`Unmodified
`
`2′-O-methyl
`ribose
`
`Phosphoro-
`thioate
`
`Compen-
`sated
`
`Mis-
`match
`
`Blunt
`
`0.5
`
`2 5 15 30 0.5 2 5 15 30 0.5 2 5 15 30
`
`5 15 30 5 15 30
`
`0.5 2 5 15 30
`
`9 nt
`
`5′ cleavage
`product (11 nt)
`
`5′ cleavage
`product (9 nt)
`
`Fig 2 | The passenger strand is cleaved during RNA-induced silencing complex assembly. (A) Graphical representation of short interfering RNAs
`(siRNAs) composed of unmodified guide strands and unmodified or chemically modified passenger strands. In all cases, the passenger strands were
`0
`phospho-radiolabelled (indicated by ‘32P’). The dotted line depicts the position where cleavage is predicted to take place on the passenger strand,
`5
`that is, between guanosine-9 (G9) and adenosine-10 (A10). The expected 9-nt cleavage product is indicated. (B) Graphical representation of an siRNA
`0
`-phospho-radiolabelled, unmodified passenger strand (for the sequence, see ‘mismatch 9–12’ in the legend of Fig 1E) and guide strands,
`formed by a 5
`the sequence of which either compensates for the 4-nt mutation in the passenger strand or leads to a central 4-nt mismatch. The dotted line depicts
`0
`the predicted cleavage position. (C) Graphical representation of a blunt siRNA formed by an unmodified guide strand and a 5
`-phospho-radiolabelled,
`0
`end. The dotted line depicts the predicted cleavage position,
`unmodified passenger strand, in which the sequence has been shifted 2 nt towards the 5
`which has also shifted by 2 nt. The expected 11-nt cleavage product is indicated. (D) Phosphorimaging analysis of a time-course cleavage reaction
`resolved in a 15% denaturing gel electrophoresis. The region of the gel corresponding to sizes between 8 and 12 nt has been enhanced for an optimal
`visualization of the cleavage products. The picture of the whole gel is depicted in supplementary Fig S4 online.
`
`released intact from the original siRNA (supplementary Fig S3
`online). To provide conclusive evidence for the cleavage of the
`passenger strand during RISC assembly, we set out to detect the
`predicted 9-nt cleavage product. We performed a time-course
`0
`-phospho-
`analysis using HeLa cytoplasmic extracts and 5
`radiolabelled siRNAs, the passenger strands of which were left
`0
`-O-methyl ribose
`unmodified, or were modified either with a 2
`at G9 or with a phosphorothioate bond between G9 and A10
`(Fig 2A). The unmodified passenger strand yielded the expected
`9-nt cleavage product after only 5 min of incubation, reached a
`maximum at 15 min and decreased thereafter (Fig 2D, left panel).
`In sharp contrast, the modified passenger strands were not cleaved
`(Fig 2D,
`left panel). Similarly, a passenger strand featuring a
`central 4-nt mismatch (Fig 2B) failed to yield a 9-nt cleavage
`product
`(Fig 2D, central panel). However, passenger strand
`cleavage could be rescued in the latter case by restoring the
`complementarity on the guide strand (Fig 2D, central panel). It is
`worth noting that the profiles of the 9-nt cleavage product differ
`0
`0
`–5
`exonucleolytic degradation of the
`from the concomitant 3
`passenger strand, which leads to the steady accumulation of
`o21 nt species during the course of the reaction (supplementary
`
`Fig S4 online). Moreover, this unspecific degradation is present in
`all cases, irrespective of whether the passenger strand is being
`cleaved or not.
`Interestingly, the cleavage of the passenger strand correlates
`temporally with the interaction of Ago2 and the siRNA (compare
`Fig 2D with supplementary Fig S2D online). As soon as Ago2
`contacts the passenger strand of the siRNA, the 9-nt cleavage
`product emerges, supporting our hypothesis that the passenger
`strand is cleaved in the course of RISC assembly.
`It is well established that the cleavage position on a target
`0
`end of
`the guide strand
`RNA is located 10 nt
`from the 5
`(Elbashir et al, 2001b). To test whether this rule also applies
`0
`to the cleavage of
`the passenger strand, we extended the 5
`end of
`the guide strand by 2 nt, resulting in a blunt-ended
`siRNA (Fig 2C). This duplex cleaved a complementary target
`RNA with a 2-nt shift (data not shown). The cleavage site on the
`passenger strand was also shifted by 2 nt, now generating an
`11-nt cleavage product (Fig 2D, right panel). This argues that
`RISC, during assembly, cleaves the passenger strand by a
`mechanism similar to that by which functional RISC cleaves a
`complementary target RNA.
`
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`

`RISC assembly in human cells
`P.J.F. Leuschner et al
`
`scientific report
`
`C
`
`Predicted
`cleavage
`
`32P
`
`32P
`
`9-nt cleavage
`product
`
`3′
`
`5′
`
`12+9
`
`12*+9
`
`12*+9
`12+9
`1 2 5 10 1 2 5 10
`
`9 nt
`
`min
`
`D
`
`nt
`12
`
`11
`
`10
`
`9
`
`9*+10
`
`9+12
`
`10*+11
`
`10+11
`
`11*+10
`
`11+10
`
`12*+9
`
`12+9
`
`21*
`
`21
`
`E
`
`100
`
`12+9
`
`100
`
`12*+9
`
`10
`
`1
`
`12 11 10
`nt
`
`9
`
`12 11 10
`nt
`
`9
`
`10
`
`1
`
`percentage (log)
`Relative intensity
`
`21-nt guiding strand
`
`21 (10)
`21*
`(9)
`12+9
`(8)
`12*+9
`(7)
`11+10
`(6)
`11*+10 (5)
`10+11
`(4)
`10*+11 (3)
`(2)
`9+12
`(1)
`9*+10
`
`A
`(1)
`
`5′
`
`3′
`
`(2)
`
`(3)
`
`(4)
`
`(5)
`
`(6)
`
`(7)
`
`(8)
`
`(9)
`
`(10)
`
`B
`
`Passenger
`strand (nt)
`
`5′ cleavage
`product
`
`Fig 3 | Functional RNA-induced silencing complex assembles on a short interfering RNA in which an otherwise non-cleavable passenger strand is
`nicked at the putative cleavage site. (A) Graphical representation of short interfering RNAs (siRNAs) formed by one (21 nt) or two passenger strands
`(x þ y) of different lengths leading to a nicked passenger strand of 21 nt. Numbers on the right refer to the length of the strands in nucleotides. The
`0
`-O-methyl ribose at G9. (B) Phosphorimaging analysis of a cleavage reaction resolved in a 6% denaturing gel electrophoresis
`asterisk indicates a 2
`showing the efficiency of target RNA cleavage as a function of the position of a nick on the passenger strand and the presence or absence of a
`-O-methyl ribose at the cleavage position. (C) Graphical representation of siRNAs formed by two passenger strands (12 nt þ 9 nt), in which the
`0
`2
`phospho-radiolabelled (indicated by ‘32P’), and either left unmodified (duplex 12 þ 9) or modified with a 2
`0
`0
`-O-methyl ribose
`12-nt fragment has been 5
`at position G9 (duplex 12* þ 9). Numbers on the right refer to the length of the strands in nucleotides. The dotted line depicts the position where
`cleavage is predicted to take place on the passenger strand, that is, between G9 and A10. The expected 9-nt cleavage product is indicated.
`(D) Phosphorimaging analysis of a time-course cleavage reaction resolved in a 15% denaturing gel electrophoresis. A phosphorylated, 9-nt RNA
`0
`0
`oligonucleotide was used as a marker. Note the different pattern between the cleavable and non-cleavable passenger strand. Whereas unspecific 3
`-to-5
`exonucleolytic degradation of the non-cleavable passenger strand leads to the steady accumulation of o12 nt species during the course of the reaction,
`the cleavable passenger strand, which is subjected to a similar degradation, shows a clear enrichment of the 9-nt species. Arrows indicate the time
`point at which the quantification in (E) was performed. (E) Quantification of the phosphorimaging analysis in (D). Bars represent the relative intensity
`of the bands, the sizes of which are indicated along the x axis. The 9-nt cleavage product accumulates only when using the unmodified, cleavable 12-nt
`fragment of the passenger strand.
`
`Bypassing the passenger strand cleavage event
`Why does the cleavage of
`the passenger strand facilitate the
`generation of functional RISC? A nick in the passenger strand is
`expected to result in a drastic change in the thermodynamic
`profile of the siRNA duplex, which could sustain the removal
`of the passenger strand. We therefore generated an siRNA by
`annealing two passenger strands of 9 and 12 nt length to a 21-nt
`guide strand, a so-called ‘9 þ 12’, nicked siRNA (Fig 3A). This
`siRNA faithfully interacted with Ago2 (data not shown) and guided
`target RNA cleavage as efficiently as an siRNA containing a
`conventional 21-nt passenger strand did (Fig 3B, lanes 2,10). This
`
`result indicates that the Ago2-mediated cleavage event per se
`appears to be not essential for the generation of functional RISC, as
`it can be bypassed by a pre-existing nick. It thus argues against a
`model in which the cleavage event imposes a conformational
`change that would facilitate the removal of the passenger strand.
`We also generated a duplex ‘9* þ 12’, in which a 2
`0
`-O-methyl
`group was placed at the G9 position (the cleavage site) of the 9-nt
`passenger strand (Fig 3A). This duplex efficiently cleaved the target
`0
`-O-methyl group at the cleavage site
`RNA, demonstrating that a 2
`has no effect on the removal of the cleaved passenger strand
`(Fig 3B, lane 1). More importantly, the complete rescue of target
`
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`EMBO reports VOL 7 | NO 3 | 2006
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`scientific report
`
`RISC assembly in human cells
`P.J.F. Leuschner et al
`
`RNA cleavage by providing a nick on the cleavage site of an
`otherwise non-cleavable passenger strand highlights the impor-
`tance of passenger strand cleavage during RISC assembly.
`Interestingly, we found that displacing the nick stepwise further
`downstream along the passenger strand and, additionally, main-
`taining its non-cleavable character (Fig 3A, 10* þ 11, 11* þ 10,
`12* þ 9) led to a reduction in target RNA cleavage, as the distance
`0
`-O-methyl group increased (Fig 3B,
`between the nick and the 2
`lanes 3,5,7).
`In contrast, duplexes with unmodified passenger
`(Fig 3A, 10þ 11, 11 þ 10, 12 þ 9) guided efficient
`strands
`cleavage of the target RNA (Fig 3B, lanes 4,6,8), underlining the
`relevance of passenger strand cleavage at the ‘natural’ position,
`that is, the phosphodiester bond between nt 9 and 10 counting
`0
`end. An intact, 21-nt non-cleavable passenger strand
`from its 5
`showed the most pronounced impairment of target RNA cleavage
`compared with a 21-nt cleavable passenger strand (Fig 3B, lanes
`9, 10, respectively). We proposed that the inhibition of target RNA
`cleavage observed for the 12* þ 9 duplex should correlate with
`the inability to cleave the 12-nt, modified passenger strand into a
`9-nt cleavage product. Indeed, specific accumulation of the 9-nt
`cleavage product was observed only when using the unmodified
`12 þ 9 duplex and not the modified 12* þ 9 duplex (Fig 3C–E).
`These findings raise a couple of interesting questions. As a
`central 4-nt mismatch on the siRNA also impaired the removal
`of
`the passenger strand, an explanation has to be found for
`how microRNAs (miRNAs) with several internal bulges, even at
`the putative cleavage site, are loaded into microRISC (miRISC).
`Furthermore, Ago1, Ago3 and Ago4 have been shown to be
`associated with mature miRNAs (Liu et al, 2004; Meister et al,
`2004). As
`in mammals only Ago2 is known to harbour
`endonucleolytic activity, how do the catalytically inactive
`Argonaute proteins remove the passenger strand? Clearly, a
`bypass pathway should exist, which allows the assembly of
`miRISC in the absence of slicer activity. During miRNA matura-
`tion, an auxiliary factor might assist in the loading of miRISC
`independently of
`the presence of a catalytically active Ago
`protein. A different possibility might
`rely on the reduced
`thermodynamic stability of miRNA duplexes, as, in contrast to
`siRNAs, miRNA duplexes feature bulges of non-paired bases at
`different positions. Interestingly, cleavage assays carried out at
`37 1C rescued target RNA cleavage guided by an siRNA contain-
`ing a central, 4-nt mismatch on the passenger strand (Fig 4A,B). In
`contrast, siRNAs featuring fully complementary passenger strands
`0
`-O-methyl or phosphorothioate modifications at
`and showing 2
`the cleavage site showed a minor rescue (Fig 4A,B).
`Finally, we tested the effect of siRNAs containing modified
`passenger strands in vivo. We co-transfected HeLa cells with
`a plasmid containing the firefly luciferase gene together with
`siRNAs harbouring differently modified passenger strands (Fig 4C)
`and analysed luciferase activities normalized to cells transfected
`with a mock siRNA. An unmodified siRNA effectively silenced
`the reporter gene, whereas we observed a major impairment in
`0
`-O-methyl ribose
`silencing with a passenger strand containing a 2
`at position 9 (Fig 4D), certifying that cleavage of the passenger
`strand also has a role in vivo. The same modification at position
`10, however, did not affect
`the knockdown efficiency. The
`most severe impairment in silencing was caused by a passenger
`0
`-O-methyl ribose at position 9 and a
`strand containing both a 2
`phosphorothioate bond between nt 9 and 10. A passenger strand
`
`with a central mismatch at positions 9 and 10 was also very
`inefficient in silencing after 6 h. However, in contrast to all other
`modifications that impaired the knockdown, silencing improved
`slightly after 24 h, probably owing to a putative bypass mechanism.
`We also tested silencing of an endogenous gene, human
`cohesin (Scc1), using both unmodified and modified duplexes
`0
`-O-methyl ribose at position
`(Fig 4E). A passenger strand featuring a 2
`9 led to a less efficient silencing than an unmodified passenger strand
`(Fig 4F). However, after enhancing the non-cleavable character
`by adding a phosphorothioate bond between nt 9 and 10 of the
`passenger strand, a severe impairment was detected (Fig 4F).
`In conclusion, we show that the cleavage of the passenger
`strand is indeed a prerequisite for effective assembly of functional
`human RISC. Our results may also provide valuable clues for the
`findings in D. melanogaster, in which RISC assembly has been
`shown to depend on the presence of Ago2 (Okamura et al, 2004;
`Tomari et al, 2004a, b). The results from HeLa cytoplasmic
`extracts favour a model in which efficient and fast removal of
`the passenger strand is dependent on the catalytic activity of Ago2.
`We also propose that removal of the cleaved passenger strand
`during RISC assembly follows similar rules as the removal of the
`cognate target RNA after cleavage: RISC, during its assembly, has
`to be liberated from the passenger strand that would otherwise
`block the recognition of a target RNA. In turn, functional RISC, as
`every multiple turnover enzyme (Hutva´gner & Zamore, 2002;
`Haley & Zamore, 2004; Martinez & Tuschl, 2004), has to
`guarantee product release only after catalysis.
`Note: During the submission process, Matranga et al, Rand
`et al (Cell, Immediate Early Publication, 3 November 2005) and
`Miyoshi et al (Genes and Development, December 2005) reported
`a similar function of the passenger strand in Drosophila. Kraynack
`& Baker (RNA, Epub ahead of print, 21 November 2005) report
`that for some siRNA sequences, passenger strands fully modified
`0
`-O-methyl residues do not impair silencing.
`with 2
`
`METHODS
`Short interfering RNA duplexes. The sequence of the passen-
`0
`0
`-CGUACGCGGAAUACUUCGAAA-3
`. The
`ger
`strand was 5
`0
`-UCGAAGUAUUCCGC
`sequence of
`the guide strand was 5
`0
`. All duplexes contained symmetric 2-nt overhangs
`GUACGUG-3
`0
`at the 3
`end. Duplexes were annealed to a final concentration of
`10 mM in 100 mM KOAc, 2 mM Mg(OAc)2 and 30 mM Hepes (pH
`7.4), and dilutions were made thereof. Oligoribonucleotides were
`obtained from Dharmacon Research Inc. (Lafayette, CO, USA) and
`PROLIGO Primers and Probes (Paris, France) and were generously
`provided by Professor Tom Tuschl (The Rockefeller University).
`In the case of 4S-U-modified passenger strands in Fig 1C, the
`base at the positions indicated was exchanged by a 4S-U residue.
`0
`-CGU
`The sequences of the passenger strands in Fig 1E were 5
`0
`0
`for ‘mismatch 5–8’, 5
`-CGUACG
`AGCGCGAAUACUUCGAAA-3
`0
`0
`for ‘mismatch 9–12’, 5
`-CGUACGCG
`CGAGUAACUUCGAAA-3
`0
`0
`for ‘mismatch 13–16’ and 5
`-CGUACGC
`GAAUAUUCAGAAA-3
`0
`for ‘mismatch 17–19’.
`GGAAUACUUAGCAA-3
`0
`-CGUA
`The sequence of the passenger strand in Fig 2C was 5
`0
`CGCGAGUAACUUCGAAA-3
`. To restore complementarity, the
`sequence of the guide strand was changed accordingly.
`To generate the blunt siRNA duplex in Fig 2C, the sequence of
`0
`-UUUCGAAGUAUUCCGCG
`the guide strand was changed to 5
`0
`.
`UACG-3
`
`3 1 8
`
`EMBO reports VOL 7 | NO 3 | 2006
`
`&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
`
`

`

`RISC assembly in human cells
`P.J.F. Leuschner et al
`
`scientific report
`
`Passenger
`strands
`
`G9-A10-P-thioate
`
`G9-2′-O-methyl
`
`Mismatch 9–12
`
`Unmodified
`
`30
`
`37
`
`30
`
`37
`
`30
`
`37
`
`30
`
`37 Temperature (°C)
`
`3′
`
`5′
`
`Unmodified
`
`B
`
`Mismatch 9–12
`
`G9-2′-O-methyl
`
`G9-A10-P-thioate
`
`3′
`
`5′
`
`Unmodified
`
`G9-2′-O-methyl
`
`A10-2′-O-methyl
`
`G9-2′-O-methyl +
`G9-A10-P-thioate
`
`Mismatch 9–12
`
`D
`
`100
`
`6 h
`24 h
`
`80
`
`60
`
`40
`
`20
`
`Luciferase activity (%)
`
`0
`
`-2′-O-m ethyl +
`-2′-O-m ethyl
`-2′-O-m ethyl
`Un m odified
`Mism atch 9–10
`-P-thioate
`
`G 9
`
`A 10
`
`-A 10
`G 9
`G 9
`
`Passenger
`strands
`
`U9-G10-P-thioate
`U9-2′-O-methyl +
`U9-2′-O-methyl
`
`Unmodified
`
`Mock siRNA
`
`Anti-Scc1
`
`Anti-proteasome
`
`3′
`
`5′
`
`Unmodified
`
`F
`
`U9-2′-O-methyl
`
`U9-2′-O-methyl +
`U9-G10-P-thioate
`
`A
`
`C
`
`E
`
`Fig 4 | In vitro and in vivo analysis of a putative bypass mechanism for RNA-induced silencing complex assembly. (A) Graphical representation of
`short interfering RNA (siRNAs) containing unmodified or chemically modified passenger strands. (B) Phosphorimaging analysis of a cleavage reaction
`resolved in a 6% denaturing gel electrophoresis. (C) Same as (A). (D) Luciferase activities measured after 6 or 24 h after transfecting HeLa cells with
`siRNAs depicted in (C). (E) Same as (A). (F) Western blot analysis of a knockdown experiment in HeLa cells 24 h after transfecting HeLa cells with
`siRNAs depicted in (E).
`
`the duplexes used in the knockdown
`The sequences of
`experiment in Fig 4E,F were taken from Hirota et al (2004).
`The sequence of the passenger strands used in the luciferase
`0
`0
`-CUUACGCUGAGUACUUCGAAA-3
`.
`assay (Fig 4C,D) was 5
`0
`-CUUACGC
`For the duplex ‘mismatch 9–10’, the sequence was 5
`0
`. The sequence of the guide strand was
`UACGUACUUCGAAA-3
`0
`0
`-UCGAAGUACUCAGCGUAAGUG-3
`.
`5
`0
`phospho-radiolabelling
`When indicated in the figure legends, 5
`of the respective strands was performed as described previously
`(Martinez & Tuschl, 2004).
`Cleavage reactions. Cleavage reactions were performed as
`described previously (Martinez et al, 2002), with the exceptions
`that MgCl2 was used at 5 mM and that the final concentration of
`siRNAs was adjusted to 10 nM. HeLa cytoplasmic extracts were
`provided by Professor Reinhard Luhrmann (Department of Cellular
`Biochemistry, Max Planck Institute for Biophysical Chemistry,
`
`Go¨ ttingen, Germany) and by Paragon Bioservices Inc. (Baltimore,
`MD, USA). The substrate RNA is 32P-cap-labelled (Elbashir et al,
`2001b). Radiolabelled markers were generated by partial RNase
`T1 digestion of the 32P-cap-labelled RNA substrate.
`Passenger strand cleavage experiments were performed as
`cleavage reactions, the difference being the absence of 32P-cap-
`labelled substrate RNA.
`Cell culture and transfections. Cell culture and transfections
`were performed as described previously (Hirota et al, 2004).
`HeLa cells were seeded at a density of 4  104 cells per 12-well
`plate, 1 day before transfection. siRNA duplexes were used
`at a final concentration of 200 nM. At 24 h after transfection,
`the cells were washed with cold PBS and lysed directly in 1 
`SDS–polyacrylamide gel electrophoresis
`sample buffer. For
`luciferase assays, HeLa cells were cultured in 24-well plates and
`transfected with 100 nM siRNAs, 0.2 mg of pGL3 promoter (Pp-luc)
`
`&2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
`
`EMBO reports VOL 7 | NO 3 | 2006
`
`3 1 9
`
`

`

`scientific report
`
`RISC assembly in human cells
`P.J.F. Leuschner et al
`
`and 0.01 mg of phRL-TK control vector (Rr-luc; Promega, Madison,
`WI, USA)
`for internal standardization. We used sextuplicates
`for each condition. Cells were collected and extracts were
`assayed 6 and 24 h after transfection.
`Western blotting. Samples were sonicated and boiled before
`being loaded on an 8% SDS–polyacrylamide gel electrophoresis.
`Gels were blotted onto polyvinylidene difluoride membranes
`using the semi-dry method. Membranes were blocked in 3%
`non-fat dry milk in Tris-buffered saline þ 0.05% Tween. Anti-
`bodies against human cohesin (Scc1) and the proteasome were
`used at a concentration of 2 mg/ml (Sumara et al, 2002). Secondary
`antibodies were from JacksonImmunoResearch (Sigma Aldrich,
`West Grove, PA, USA).
`Supplementary information is available at EMBO reports online
`(http://www.emboreports.org).
`
`ACKNOWLEDGEMENTS
`We thank A. Kuras, G. Obernosterer, D. Pezic and S. Weitzer, members
`of the laboratory, for encouragement and suggestions during the
`completion of this work, Professor R. Schroeder and C. Ribeiro for
`critically reading the manuscript, H. Manninga for the synthesis of
`4-thio-uridine-modified siRNAs and for comments on the manuscript and
`Y. Dorsett for discussions. J.M. is a Junior Group Leader at IMBA.
`P.J.F.L. is funded by the Boehringer Ingelheim Fonds PhD Scholarship
`and S.L.A. is funded by the Vienna Biocenter PhD program and partly by
`Fonds zur Fo¨ rderung der Wissenschaftlichen Forschung through WK001.
`IMBA is the Institute of Molecular Biotechnology supported by the
`Austrian Academy of Sciences.
`
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