Endogenous Nuclear RNAi Mediates
Behavioral Adaptation to Odor
Bi-Tzen Juang,1Chen Gu,1,2Linda Starnes,1,3Francesca Palladino,4Andrei Goga,1Scott Kennedy,5
and Noelle D. L’Etoile1,*
1Departments of Cell & Tissue Biology and Medicine, University of California, San Francisco, 513 Parnassus Avenue, San Francisco,
CA 94143-0512, USA
2Amunix, Inc., 500 Ellis Street, Mountain View, CA 94043, USA
3Chromatin Structure and Function Group, NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen,
Blegdamsvej 3B, Room 4.3.07, 2200 Copenhagen N, Denmark
4E´cole Normale Supe ´rieure de Lyon, CNRS, Molecular Biology of the Cell Laboratory/ UMR5239, Universite ´ Claude Bernard Lyon,
69007 Lyon, France
5Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA
Most eukaryotic cells express small regulatory
RNAs. The purpose of one class, the somatic endog-
enous siRNAs (endo-siRNAs), remains unclear. Here,
we show that the endo-siRNA pathway promotes
odor adaptation in C. elegans AWC olfactory neu-
rons. In adaptation, the nuclear Argonaute NRDE-3,
which acts in AWC, is loaded with siRNAs targeting
odr-1, a gene whose downregulation is required for
adaptation. Concomitantwithincreased odr-1siRNA
in AWC, we observe increased binding of the HP1
homolog HPL-2 at the odr-1 locus in AWC and
reduced odr-1 mRNA in adapted animals. Phosphor-
ylation of HPL-2, an in vitro substrate of the EGL-4
kinase that promotes adaption, is necessary and
sufficient for behavioral adaptation. Thus, environ-
mental stimulation amplifies an endo-siRNA negative
feedback loop to dynamically repress cognate gene
expression and shape behavior. This class of siRNA
may act broadly as a rheostat allowing prolonged
stimulationto dampen gene expression and promote
cellular memory formation.
RNA interference (RNAi) has beenexploited asapowerful exper-
imental tool in both somatic and germ cells for over a decade
(Fire et al., 1998), and organisms ranging in complexity from
yeast to humans produce a range of endogenous small RNAs
of 20–30 nucleotides in length. Although it is apparent that
almost all cells of an organism are actively engaged in some
form of endogenous RNAi, its role, particularly in somatic
cells, remains unclear (reviewed in Ketting, 2011; Ghildiyal and
Endogenous small RNAs are grouped into three classes
according to their biosynthetic origin and the proteins they
bind: piwi-RNAs (piRNAs), micro RNAs (miRNAs), and endoge-
are encoded by genes, whereas in C. elegans, endo- siRNAs are
produced by RNA-dependent RNA polymerases that use
thousands of cellular messenger RNAs (mRNAs) as templates
to produce antisense small RNAs (Ghildiyal and Zamore, 2009;
Ketting, 2011; Gent et al., 2010; Gu et al., 2009). Small RNAs
have been linked to synaptic function and memory formation in
mammals (McNeill and Van Vactor, 2012). For instance, the
microRNA miR134 was shown to repress context-dependent
fear learning and long-term potentiation in mice (Gao et al.,
2010), and a piRNA has been shown to promote long-term
synaptic facilitation of cultured Aplysia sensory neurons (Rajase-
thupathy et al., 2012). However, the extent to which small RNAs
couple environmental stimuli to synaptic plasticity and the
mechanism by which small RNAs regulate experience-induced
behavioral changes remain a mystery.
Prolonged odor exposure induces a form of behavioral plas-
ticity termed adaptation. C. elegans is innately attracted to
food-related odors, but the attraction is diminished if starvation
accompanies the odor. The resulting odor-adapted state lasts
until the animal is fed (Colbert and Bargmann, 1997; Lee et al.,
2010). Odor sensation (Bargmann et al., 1993) and adaptation
(L’Etoile et al., 2002) occur within the olfactory sensory neuron
that is referred to as AWC. Whereas odor sensation requires
the guanylyl cyclase (GC) ODR-1, odor adaptation requires
downregulation of ODR-1 (L’Etoile and Bargmann, 2000).
Decreased intracellular cGMP, in part, drives the cGMP-depen-
dent protein kinase EGL-4 into the AWC nucleus (O’Halloran
et al., 2012). Once in the nucleus, EGL-4 is both necessary and
sufficient to induce long-lasting odor adaptation (Lee et al.,
2010). The mechanism by which nuclear EGL-4 triggers long-
lasting odor adaptation is not known.
Small RNAs can regulate gene expression in both the cyto-
plasm and nucleus. For instance, miRNAs and siRNAs act as
guides to target mRNAs for repression in the cytoplasm (re-
viewed in Ketting, 2011; Ghildiyal and Zamore, 2009). piRNAs
and siRNAs can enter nuclei to trigger cotranscriptional gene
silencing (nuclear RNAi) (Guang et al., 2008; Le Thomas et al.,
1010 Cell 154, 1010–1022, August 29, 2013 ª2013 Elsevier Inc.
2013). During nuclear RNAi in C. elegans, the Argonaute (Ago)
transcripts that exhibit sequence complementarity to NRDE-3-
associated siRNAs (Guang et al., 2008; Guang et al., 2010).
NRDE-3 recruits the conserved nuclear protein NRDE-2 and
NRDE-4, to RNAi-targeted nascent transcripts to inhibit RNA
polymerase II (RNAP II) elongation (Guang et al., 2010; Burkhart
et al., 2011). In addition, genes targeted for silencing by the
nuclear RNAi pathway accumulate the repressive chromatin
mark, H3K9me3 (Guang et al., 2010; Burton et al., 2011). In the
C. elegans germline, piRNAs and siRNAs trigger nuclear RNAi
at thousands of genomic loci (Claycomb et al., 2009; Gu et al.,
2009; Ashe et al., 2012; Lee et al., 2012; Shirayama et al.,
2012), and the silencing effects can endure for more than five
generations (Vastenhouw et al., 2006; Buckley et al., 2012).
When nuclear RNAi is disabled, C. elegans germlines lose their
immortal character (Buckley et al., 2012).
of evidence indicate that, in the AWC olfactory sensory neurons
of adult-behaving C. elegans, endogenous RNAi promotes
odor adaptation by repressing the odr-1 gene. First, we show
cipitates (coIPs) odr-1-directed endo-siRNAs, and in adapted
Third, odor exposure diminishes the levels of odr-1 mRNA.
Fourth, in odor adaptation, HPL-2, a heterochromatin-binding
protein, is loaded onto the odr-1 locus. Additionally, we find
that phosphorylation of HPL-2 at sites that are in vitro targets
of the odor-responsive kinase EGL-4 is both necessary and
sufficient to promote odor adaptation in the AWC neurons of an
mentally relevant experiences may regulate gene expression,
thereby shaping behavior in a specific and dynamic fashion.
The Nuclear RNAi Argonaute NRDE-3 Is Required in the
AWC Sensory Neuron for Odor Adaptation
C. elegans is innately attracted to the odor, butanone. Attraction
is assessed by the chemotaxis assay shown in Figure 1A, which
allows quantification of the behavior in the form of a chemotaxis
index (CI) (Bargmann et al., 1993). Naive wild-type animals
exhibit a high CI to butanone, which decreases after 80 min of
butanone exposure in the absence of food (Colbert and
Bargmann, 1995). This experience-dependent decrease in CI is
termed long-term olfactory adaptation. If the adapted CI is
greater than one half of the naive CI, a strain is considered
To investigate the role of small RNAs in long-term olfactory
adaptation, we examined butanone adaptation in strains defec-
tive for major pathways producing RNAi in the soma, including
the microRNA, exogenous RNA (exo-RNAi), and endogenous
RNAi pathways. Animals lacking Dicer (DCR-1) were defective
for adaptation (Figure 1B). Dicer, an RNAase III, processes dou-
ble-stranded (dsRNA) into small noncoding RNAs (Grishok et al.,
the microRNA, exo-, and endo-siRNA interference pathways
(Grishok et al., 2001; Knight and Bass, 2001; Grishok et al.,
2005). These data suggest that Dicer-mediated processing of
dsRNA is required for adaptation. By contrast, the adapted CI
of strains bearing mutations in the pri-miRNA-processing RNase
III enzyme Drosha, DRSH-1 (Denli et al., 2004), the miRNA-
binding Ago, ALG-2 (Vasquez-Rifo et al., 2012), or the exo-
RNAi pathway Ago, RDE-1 (Tabara et al., 1999), were not
significantly different from the CI of wild-type controls (Figures
1B and S2 available online). These data suggest that, if
Dicer-mediated dsRNA processing is required for butanone
adaptation, microRNAs or the exoRNAi pathway are unlikely to
mediate this process.
MUT-7, a putative 30to 50exonuclease, is required for accu-
mulation of endogenous 22 nucleotide siRNAs that bind the
WAGO clade of Agos (Yigit et al., 2006; Lee et al., 2006; Gu
et al., 2009) and accumulation of 26 nucleotide siRNAs (Zhang
et al., 2011), as well as transposon and transgene silencing,
exogenous RNAi, and proper chromosome segregation (Ketting
etal.,1999;Tabaraetal.,1999;Dernburg etal.,2000;Tops etal.,
2005). MUT-7 is also required for nuclear accumulation of
NRDE-3 (Guang et al., 2008). HPL-2 is one of two C. elegans
homologs of Heterochromatin Protein 1 (HP1) (Couteau et al.,
2002). HPL-2 is involved in multiple cellular events, including
gene regulation and DNA replication and repair (Couteau et al.,
2002; Coustham et al., 2006; Black and Whetstine, 2011), as
well as transgene silencing and piRNA-mediated gene silencing
in the gonad (Grishok et al., 2005; Burkhart et al., 2011; Ashe
et al., 2012; Buckley et al., 2012; Shirayama et al., 2012). Strains
that lacked MUT-7 or HPL-2 were defective for butanone adap-
tation (Figure 1B). These results suggest that heterochromatin
and possibly small RNAs promote odor adaptation downstream
Using mut-7 and hpl-2 promoter fusions to drive expression of
GFP-tagged MUT-7 or HPL-2, respectively, we observed GFP
expression in many cells, including both AWCs (Figure 1C). To
determine whether MUT-7 and HPL-2 act in the AWC neurons,
the site of odor sensation and adaptation, we asked whether
cell-specific expression of MUT-7 and HPL-2 could rescue the
odor adaptation defect of each corresponding mutant strain.
Expressing MUT-7 or HPL-2 solely within the AWC neurons
(from the AWC-specific ceh-36prom3promoter [Etchberger
et al., 2007]) of the respective mutant strain rescued its adapta-
tion defects (Figure 1D). These data indicate that MUT-7 and
HPL-2 act within AWC neurons to promote odor adaptation.
These factors could be required at the time of odor exposure
or developmentally. To distinguish between these possibilities,
we used the heat shock promoter phsp-16.2 (Stringham et al.,
1992) to express each factor in the adult immediately prior to
odor exposure. Heat-shock-driven expression restored adapta-
tion to the mut-7 and hpl-2 strains (Figure 1E). Consistent with a
requirement in the adult, neither morphology nor cell fate of the
AWC was altered by loss of HPL-2 or MUT-7 (Figure S1B and
Table S1). Together, these results indicate that the adaptation
defects of mut-7- and hpl-2-deficient animals are not due to
To address whether MUT-7 and HPL-2 act in the same
molecular pathway, we created mut-7;hpl-2 and control
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