Protection from Feed-Forward
Amplification in an Amplified
Julia Pak,1Jay Mahesh Maniar,2,3Cecilia Cabral Mello,1and Andrew Fire1,2,*
1Department of Pathology
2Department of Genetics
Stanford University School of Medicine, Stanford, CA 94305, USA
3Present address: McKinsey and Company, Silicon Valley, Palo Alto, CA 94304 USA
The effectiveness of RNA interference (RNAi) in
many organisms is potentiated through the signal-
amplifying activity of a targeted RNA-directed RNA
polymerase (RdRP) system that can convert a small
fragments into an abundant internal pool of small
interfering RNA (siRNA). As for any biological amplifi-
cation system, we expect an underlying architecture
that will limit the ability of a randomly encountered
trigger to produce an uncontrolled and self-esca-
lating response. Investigating such limits in Caeno-
rhabditis elegans, we find that feed-forward amplifi-
cation is limited by biosynthetic and structural
distinctions at the RNA level between (1) triggers
that can produce amplification and (2) siRNA prod-
ucts of the amplification reaction. By assuring that
initial (primary) siRNAs can act as triggers but not
templates for activation, and that the resulting
(secondary) siRNAs can enforce gene silencing on
additional targets without unbridled trigger amplifi-
cation, the system achieves substantial but funda-
mentally limited signal amplification.
Canonical RNAi is a biochemical pathway triggered by foreign
dsRNA that ultimately results in the destruction of endogenous
target RNA of corresponding sequence (reviewed in Boisvert
and Simard, 2008). The core RNAi factors that initiate this
process center on Dicer (DCR-1 in Caenorhabditis elegans),
along with its dsRNA-binding partner (RDE-4 in C. elegans);
these factors start the pathway by processing the dsRNA trigger
into short interfering RNAs (siRNAs) (Knight and Bass, 2001;
Grishok et al., 2001; Tabara et al., 2002). The double-stranded
primary siRNAs have structures characteristic of RNase III type
cleavages, 2 nt 30overhangs, 30hydroxyls (30-OH), and 50mono-
phosphates (50-monoP) (Elbashir et al., 2001a, 2001b, 2001c).
Primary siRNAs are then transferred to Argonaute class RNA-
binding proteins, with cleavage of one strand leaving a single-
sequences in a target RNA pool. Argonautes possess three
main domains: the PAZ domain, which binds the 30terminus of
the siRNA; the MID domain, which binds the 50terminus of the
siRNA; and the PIWI domain that folds into an RNase H-like
structure (Song et al., 2004). In a number of systems, most
notably inmammalsandDrosophila,thedestruction ofthetarget
RNA has been shown to be mediated by a cleavage activity of
the Argonaute RNase H domain, guided by the bound siRNA
(reviewed in Nowotny and Yang, 2009). Although the simplicity
of this canonical RNAi paradigm provides some indications of
its potential biological effect, it appears from the stoichiometry
in several systems that a simple one-siRNA-one-target relation-
ship would be insufficient for the degree of gene silencing that is
How does the RNAi pathway ensure robust silencing of target
RNAs? One solution relies on the mechanistic ability of individual
RNA-induced silencing complex (RISC) assemblies to serially
target multiple mRNAs (Hutva ´gner and Zamore, 2002). In some
organisms such as fungi, plants, and C. elegans, the potentially
multiturnover core RNAi mechanism is supplemented through
the action of RdRP that expand the initial siRNA pool with the
generation of secondary siRNAs (Cogoni and Macino, 1999;
Mourrain et al., 2000; Sijen et al., 2001). (Although Drosophila
and mammals do not appear to possess canonical RdRPs, the
latter may use other polymerases to perform RNAi-mediating
RdRP function; e.g., Lehmann et al., 2007; Maida et al., 2009).
The C. elegans genome encodes four putative RdRPs: RRF-1,
?2, ?3, and EGO-1. While rrf-1 and ego-1 were initially
shown to be required for RNAi in the soma and germline, respec-
tively, rrf-3 appears to mediate endogenous gene-silencing
functions and rrf-2’s function(s) has yet to be discovered (Sijen
et al., 2001; Simmer et al., 2002; Smardon et al., 2000; Gent
et al., 2009; Pavelec et al., 2009; Vasale et al., 2010). Current
in RNAi: a ‘‘primary’’ pool produced upon action of Dicer on the
initial dsRNAtriggerwithadditional smallRNAproducedthrough
targeted activity of the RdRPs. Previous work has illustrated
a potentially important structural distinction between primary
can engage complementary
Cell 151, 885–899, November 9, 2012 ª2012 Elsevier Inc. 885
siRNAs and RdRP products in that the latter often retain the 50
triphosphate (50-PPP) from the initiating nucleotide (Pak and
Fire, 2007; Sijen et al., 2007).
Biological effects of 20–30 nt RNAs in eukaryotes frequently
involve their incorporation into RISCs that include a member of
the Argonaute protein family whose sequence-directed activity
is programmed by a single small RNA effector. The C. elegans
genome encodes 27 putative Argonautes, a diversity supporting
the varied biological activities and molecular roles of several
small RNA families in this organism, including microRNAs,
piRNAs, and several types of endogenous siRNAs (Grishok
et al., 2001; Yigit et al., 2006; Guang et al., 2008; reviewed in
Fischer, 2010). Small RNAdistributions duringan RNAi response
in C. elegans further exemplify the potential for mechanistic
plurality inherent in a diversity of Argonautes, with 50-monoP
(primary) siRNAs having been shown to bind the Argonaute
RDE-1, whereas 50-PPP siRNAs (RdRP products) bind the
‘‘WAGO’’ group of worm-specific Argonautes (Yigit et al., 2006).
Current models posit the expansion of small RNA pools during
RNAi as resulting from an ability of individual siRNA RISC
complexes interacting with a target message to recruit RdRP
and thereby generate a target-limited population of RdRP
products. This model, involving four types of RNA species (initial
dsRNA trigger, target mRNA, primary siRNAs, and RdRP prod-
ucts), certainly allows for amplification and propagation of
RNAi responses as long as populations of trigger and target
RNAs are present.
Given the efficacy of RdRP activity, why don’t RNAi processes
amplify indefinitely (Sijen et al., 2001; Bergstrom et al., 2003; Pak
and Fire, 2007; Sijen et al., 2007)? Unbridled secondary siRNA
generation could potentially create havoc, generating diverse
sequences that could potentially direct the destruction of unre-
lated RNAs. Experimental analysis of RNAi in C. elegans offers
the flexibility of altering the structure and delivery of trigger
RNA as well as the genetic background of the recipient animals
in characterizing the interference response. Combining these
two capabilities with high-throughput sequencing to charac-
terize populations ofRNAs associated withRNAi, wehaveinves-
tigated the mechanistic basis for limitations to amplification of
RNAi in C. elegans.
Properties of Primary and Secondary siRNA Pools
In C. elegans, RNAi proceeds in two phases: the primary
and secondary siRNA responses. To distinguish between these
phases, we established an assay wherein RNAi against the sel-1
gene was initiated by feeding the animals with a dsRNA trigger
(Timmons et al., 2001) that contained a series of mismatches
from the wild-type sequence at 25 nt intervals (Figure 1A). This
allows a sequence-based distinction between trigger-derived
RNA sequences and sequences derived from copying of target
Sequencing was used to detect sel-1 siRNAs and determine
their identity with the original target and trigger sequences.
We employed two small RNA (sRNA) capture protocols for
high-throughput sequencing using the Illumina platform. To
examine the broadest spectrum of siRNAs, we used a 50-P-inde-
pendent protocol, which does not distinguish between different
50phosphoforms (Gent et al., 2010). A second protocol that
depends on ligation to a 50-monoP should enrich for 50-monoP
species such as primary DCR-1 products (Lau et al., 2001). As
previously observed (Pak and Fire, 2007), RNAs retaining a
50-PPP terminus (such as direct RdRP products) would be
depleted in the 50-monoP-enriched pool. All of the protocols
used for library production rely on a dephosphorylated 30-OH
6,420,803 captured RNAs, of which 159,424 (2.5%) corre-
sponded to sel-1 sequence (Table 1, Figure 1B). As a control,
we examined 25,109,432 sequences compiled from multiple
sets of animals that had no exposure to sel-1 dsRNA (Gent
et al., 2010; Maniar and Fire, 2011; Wu et al., 2011); from this
gesting that the observed exogenously triggered sel-1 siRNAs in
the RNAi experiments are indeed specific to the RNAi response.
siRNAs that match the mismatched sel-1 dsRNA trigger
accounted for only 5% of total sel-1 siRNAs in the responding
animals. We found that the sense:antisense ratio of trigger-
matched siRNAs was close to 1 (0.88 and 0.93, respectively, in
the 50-monoP-enriched and 50-P-independent pools). This
contrasts with the sense:antisense ratios for the remaining
(target-matched) sel-1 siRNAs (0.00014 and 0.00284, respec-
tively) and argues that the trigger-matched population is indeed
a defined siRNA class (Tables 1 and 2). A priori, at least three
classes of RNA molecules may comprise this trigger-matched
pool: (1) fragments of RNA generated in the bacteria fed to the
animals (potentially with diverse 50structures), (2) primary
siRNAs generated directly through DCR-1-mediated processing
of dsRNA trigger molecules (with 50-monoP termini), and (3)
products of RdRP copying of the trigger RNA in C. elegans
(possessing 50-PPP termini). Although we cannot exclude the
presence of any of the aforementioned classes of RNAs, our
data are consistent with a substantial fraction of the trigger-
matched siRNA population carrying a 50-monoP. In particular,
enriching for 50-monoP-sRNAs produced a 12-fold enrichment
for trigger-matched siRNAs among total sel-1 siRNAs (p =
0.0189) (compare N2 trigger-matched siRNAs in Tables 1 and
2). Although consistent with the 50structures of DCR-1 products,
these data do not address whether these trigger-matched prod-
uctsarederivedfromthe initial dsRNAinoculum(Bernstein etal.,
2001) or from copies of the original inoculum produced by an
We found that the majority of the RNAi-induced sel-1 siRNAs
were (1) antisense to the target mRNA, (2) corresponding to
sequence within the targeted region of the mRNA (nucleotides
535 to 992 of the mRNA; Table 1, Figure 1D), and (3) perfectly
matched to the target RNA, without the mismatches introduced
in the trigger (Figure 1D). Characterization of the target-matched
siRNAs revealed distinct length and sequence composition
proclivities, with a consensus length of 22 nt and frequent
appearance of a G at the 50end (Figure S1). (The 50G sequence
bias was also detected, to a lesser extent, in other classes of
886 Cell 151, 885–899, November 9, 2012 ª2012 Elsevier Inc.
Figure 1. Characterization of Primary and Secondary siRNA Pools with a Mismatched Trigger
(A) Mismatched trigger assay.
(B) Proportions of distinct small RNA populations from 50-P-independent small RNA capture from N2 (wild-type) animals fed mismatched sel-1 dsRNA.
(C) Proportions of distinct small RNA populations from 50-monoP-enriched small RNA capture from N2 (wild-type) animals fed mismatched sel-1 dsRNA.
(D and E) Plot of sel-1 siRNAs from 50-P-independent capture from N2 animals fed mismatched sel-1 dsRNA (D). Plot of sel-1 siRNAs from 50-monoP-enriched
capture from N2 animals fed mismatched sel-1 dsRNA(E). Gray: sel-1 mRNA, green: region of sel-1 mRNA encompassed in mismatched trigger, black: siRNA
matching both target and trigger RNAs, blue: siRNA matching only target RNA, red: siRNA matching only trigger RNA. Lightened shading indicates multiple
incidence. (See also Figure S1.)
Cell 151, 885–899, November 9, 2012 ª2012 Elsevier Inc. 887
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