A 30UTR Pumilio-Binding Element
Directs Translational Activation in
Olfactory Sensory Neurons
Julia A. Kaye,1Natalie C. Rose,5Brett Goldsworthy,6Andrei Goga,4and Noelle D. L’Etoile2,3,*
1Cellular and Developmental Biology Program, 1 Shields Drive, University of California, Davis, Davis, CA 95616, USA
2Center for Neuroscience, University of California, Davis, 1544 Newton Ct., Davis, CA 95616, USA
3Department of Psychiatry and Behavioral Sciences, 2230 Stockton Boulevard, Sacramento, CA 95817, USA
4Department of Medicine, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA
5Present address: University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA
6Present address: Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
Prolonged stimulation leads to specific and stable
changes in an animal’s behavior. In interneurons,
this plasticity requires spatial and temporal control
translational control occurs in sensory neurons is
not known. Adaptation of the AWC olfactory sensory
neurons of C. elegans requires the cGMP-dependent
protein kinase EGL-4. Here, we show that the RNA-
binding PUF protein FBF-1 is required in the adult
AWC for adaptation. In the odor-adapted animal, it
increases translation via binding to the egl-4
30UTR. Further, the PUF protein may localize transla-
RNA-binding PUF proteins have been shown to
promote plasticity in development by temporally
andspatially repressingtranslation,thiswork reveals
that in the adult nervous system, they can work in
a different way to promote experience-dependent
plasticity by activating translation in response to
Complex behaviors require accurate and efficient sensing of the
environment as well as the ability to ignore persistent stimuli that
offer little new information. Since sensory neurons provide the
information from which most behaviors arise, the ability of these
neurons to modulate their excitability, as a function of experi-
ence is critical. While brief stimulation elicits rapidly reversible
adjustments in excitability that allow the animal to track changes
in its environment, prolonged stimulation elicits more enduring
homeostatic changes that allow the animal to reset its sensitivity
to long-lasting alterations such as day-to-night changes in light
intensity (Calvert et al., 2006) or the presence of a persistent
odor (Mashukova et al., 2006; Zufall and Leinders-Zufall, 1997).
Repeated experiences also elicit changes in behavior that
require alterations in the sensitivity of interneurons within neural
circuits. Repeated stimulation or prolonged alteration in the
pattern of stimulation of specific connections can lead to long-
term potentiation (LTP), long-term depression (LTD), or homeo-
static synaptic scaling within the synaptic connections between
hippocampal cells. These changes have been shown to underlie
the animal’s ability to form memories as a function of repeated
experience (see Sutton and Schuman, 2005, 2006, for review).
The nematode C. elegans also adjusts its behavior as a func-
tion of experience. Though some odors are inherently attractive
to naive C. elegans, they will begin to ignore such odors after
etal., 1993).C.elegans isinherently attracted to over 60different
volatile chemical compounds, many of which stimulate the
paired AWA and AWC olfactory sensory neurons (Bargmann
et al., 1993). Remarkably, though each AWC can probably sense
tens of odors (Troemel, 1999), sensitivity to individual odors can
be decreased independently (Colbert and Bargmann, 1995).
Sensitivity to an odor decreases as the length of prior exposure
to that odor increases (Colbert and Bargmann, 1995; L’Etoile
et al., 2002). That is, while initial exposure to an odor gradient
elicits chemotaxis to the point source, a 30 min pre-exposure
to the same odor will reversibly decrease chemotaxis (short-
term adaptation) and exposures of more than 1 hr will stably
decrease odor-seeking for hours (long-term adaptation) (Colbert
and Bargmann, 1995). Odor sensation is thought to occur within
protein-coupled receptors reside (Sengupta et al., 1996; Troe-
mel et al., 1995). Odor sensation requires the G-alphas ODR-3
and GPA-2 (Roayaie et al., 1998; Lans et al., 2004) and is likely
to decrease cGMP. cGMP is produced by the membrane gua-
nylyl cyclases DAF-11 and ODR-1 (Birnby et al., 2000; L’Etoile
and Bargmann, 2000). Lowered cGMP levels favor closing of
the cyclic nucleotide-gated sensory channel TAX-2/4 (Chalasani
et al., 2007).
How do these sensory neurons alter their responsiveness as
a function of prolonged or repeated experience? Retinal cells
within the visual system shuttle previously synthesized adapta-
tion-promoting proteins into the sensory compartment to
dampen the incoming signal. Prolonged exposure of rods to
bright light induces massive translocation of arrestin from the
inner segment to the outer segment where arrestin binds to the
Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc. 57
Figure 1. The egl-4 30UTR Contains a Composite Pumilio and FBF Binding Site (NR/FBE) Required for Olfactory Adaptation
(A) Adaptation paradigm.In the chemotaxis assay (left), naive worms placedon an agar-lined Petri dish leave their initial location (origin) to seek an attractiveodor
source(red Xon right sideof dish).Intheadaptation assay(right), worms havebeen exposed totheodordiluted intobuffer for anhour, and theynolonger seekits
(B) Adaptation to all three AWC sensed odors was impaired by two mutations within the egl-4 30UTR; egl-4 (ky95) (L’Etoile et al., 2002).
(C) Sequence alignment of Pumilio and FBF-1 binding sites with the egl-4 NR/FBE. Target NREs and FBEs are aligned starting with the UGU, and the bases
mutated in ky95 are shown in bold red. Both alignments of the egl-4 NR/FBE with the hunchback NRE are shown.
(D) Gel mobility shift assays of the putative response element within wild-type (left top and bottom panels) and ky95 mutant (right, top and bottom) 30UTRs by
either purified GST- Drosophila Pumilio (PUM-HD) (top panels) or purified GST-FBF-1 (bottom panels). The free probe and shifted complexes are indicated
with arrows. 100 fMoles of32P-labeled 30-mer RNA probes were incubated with increasing amounts of protein for 2 hr before being gel resolved. 0.1, 1, 10,
20, 40, 80, or 160 nM of PUM-HD was incubated with the wild-type probe (top, left) while 0.1, 1, 10, 20, 40, 80, 16, 320, or 640 nM was incubated with the
‘‘ky95’’ probe (top, right). 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 40, 80, 160, or 260 nM FBF-1 was incubated with either the wild-type (bottom, left) or ‘‘ky95’’ (bottom,
A PUF Activates Translation and Sensory Adaptation
58 Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc.
GPCR rhodopsin and limits the light-induced signaling (Calvert
etal.,2006).Mammalian olfactory neuronsadjust their sensitivity
by relocating previously synthesized proteins away from
possible stimulation: prolonged odor exposure induces clathrin-
mediated endocytosis to remove the olfactory receptor GPCRs
from the cell surface (Mashukova et al., 2006). Interneurons,
however, use a different strategy to adjust to repeated stimula-
tion: they synthesize new proteins such as CamKII (Miller et al.,
2002) and Arc/Arg3.1 (Moga et al., 2004) or sensorin (Lyles
et al., 2006) within the vicinity of the stimulated synapse. The
net effect of these proteins is to alter channel composition
(Chowdhury et al., 2006) or dendritic morphology (Lyles et al.,
2006) at the stimulated synapse and thereby adjust the strength
of the synaptic connection as a function of experience. Though
translational control mechanisms are central to the function of
interneurons it is not known whether sensory neurons employ
a similar strategy.
In the C. elegans AWC sensory neurons, both short-term and
long-term adaptation require the cGMP-dependent protein
kinase EGL-4 (L’Etoile et al., 2002). Surprisingly, mutations
within the egl-4 30UTR interfere with adaptation (L’Etoile et al.,
2002). We found that these residues are part of a highly
conserved translation-repressing Nanos Response Element
(NRE) (Murata and Wharton, 1995; Zamore et al., 1997). Thus,
asin interneurons,translational controlwithin in sensoryneurons
may be crucial for appropriate behavior in the adult animal.
NREs bind the Pumilio/Fem-3 binding factor (PUF) family of
translational repressors. The founding member, Pumilio, first
discovered in Drosophila, was shown to establish the anterior-
posterior embryonic axis during development (Lehmann and
Nusslein-Volhard, 1991) by binding to and repressing translation
of maternal hunchback mRNA (Murata and Wharton, 1995).
PUFs have been characterized as translational repressors that
seem to work either by reducing translation of the target
message or increasing its decay (Wickens et al., 2002).
Biochemical evidence suggests that yeast, human, and C. ele-
gans PUFs may accomplish this by interacting with deadenylat-
ing and decapping protein complexes; (Goldstrohm et al., 2006,
2007). However, in Xenopus oocyte maturation, PUF-binding
elements in combination with cytoplasmic polyadenylation sites
have been shown to increase translation of their targets (Pique ´
et al., 2008) while in the developing hypodermis and nerve
cord of C. elegans they repress translation in combination with
microRNA-binding sites (Nolde et al., 2007). It is not known
how Pumilios either by themselves or in combination with other
factors act in the context of an adult neuron.
In other systems, PUFs have been shown to focus translation
spatially and temporally: in yeast, Puf6p restricts Ash1p expres-
sion to the bud cell by localizing ASH1 mRNA to the bud tip (Gu
et al., 2004), and in C. elegans, FBF-1/2 restrict the expression of
the sperm-promoting factor FEM-3 to the larval period when
spermatocytes are produced (Zhang et al., 1997). The ability of
PUFs to focus translation in a spatiotemporal fashion makes
them attractive candidates for promoting plasticity within
neurons. Indeed, in neurons within the developing Drosophila
larva, Pumilio’s ability to repress translation is required for
regulation of neuronal membrane excitability (Mee et al., 2004)
and appropriate synaptic development and plasticity (Menon
et al., 2004) as well as proper dendritic branching (Ye et al.,
2004). In the adult fly, dPUM is required for olfactory associative
learning (Dubnau et al., 2003) and to limit motor neuron excit-
ability (Schweers et al., 2002). In adult mice, Pumilio1 mRNA
(Rouhana et al., 2005) and PUM2 protein (Xu et al., 2007) are
observed in the hippocampus. However, the mechanisms by
which PUFs function and the targets they regulate in the adult
nervous system are not known.
Here, we show that both the PUF-binding site within the egl-4
30UTR and the PUF FBF-1 are required at the time of odor expo-
sure to promote adaptation to the odors butanone and isoamyl
alcohol. The ability of the egl-4 30UTR to bind FBF-1 is required
both for adaptation and increased expression of the egl-4
30UTR-dependent Kaede and luciferase reporters. The expres-
sion pattern of the Kaede reporter in the intact animal reveals
that FBF-1 acts via the PUF-binding site to perhaps increase
translation near the AWC sensory cilia and cell body. The lucif-
erase reporter shows that odor exposure induces a dynamic
increase in translation that is dependent upon the NR/FBE.
Thus, we describe here a novel function for a PUF-binding
element. Instead of repressing translation, as was seen in devel-
oping tissues, we find that in the adult functioning sensory
neurons FBF-1 activates translation of its target in response to
stimulation. The mechanism of activation requires a specific
sequence within and surrounding the binding site to direct
FBF-1 to increase translation and it does this without affecting
The egl-4 30UTR Contains a Novel NRE that Is Required
for Olfactory Adaptation
to an odor resulting from prolonged exposure to that odor
(Figure 1A; Colbert and Bargmann, 1995). An unbiased genetic
screen to isolate key players in the adaptation process identified
the mutant, egl-4(ky95) (L’Etoile et al., 2002). The egl-4(ky95)
mutant strain was defective for adaptation to all AWC-sensed
odors we tested (Figure 1B; L’Etoile et al., 2002). Interestingly,
the coding region of this allele of egl-4 was intact, instead we
found two point mutations in its 30UTR (L’Etoile et al., 2002)
that disrupted a putative NRE (Figure 1C). This indicated that
regulation of EGL-4 translation might be important for its
function. Points of regulation could include: basal levels of
EGL-4, its spatial distribution, or its dynamic expression in
response to prolonged odor-stimulation.
The egl-4 NRE Can Bind Both FBF-1
and Pumilio Proteins
To understand how EGL-4 expression might be regulated, we
examined the egl-4 NRE. NREs are defined by their ability to
bind the RNA binding Pumilio class of translational repressors
right) probe. TheKdsbelow eachgelweregenerated fromthreeseparategel-shiftassaysusingnonlinear regression analysis (GraphPadPrism)softwaretofitthe
percentage probe bound to sigmoidal dose response (variable slope) curves. Error bars indicate the SEM from at least three separate gel shifts.
A PUF Activates Translation and Sensory Adaptation
Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc. 59
(Zamore et al., 1997). The Drosophila genome encodes a single
PUF dPum while the C. elegans genome encodes 11 predicted
PUFs (Wickens et al., 2002) along with the newly predicted
WBGene00014165;class = Gene). The C. elegans PUF family
tree has two branches; PUF-8 and PUF-9 are closest to dPUM
and the mammalian PUFs, while the other members are on
a separate branch (Wickens et al., 2002). Members of each
branch bind to distinct RNA sequence elements: dPUM and
PUF-8/9 bind an NRE while FBF-1 and FBF-2, two members of
et al., 2005). Bernstein et al. (2005) showed that dPUM cannot
bind an FBE site conversely; FBF-1 does not interact with the
hunchback NRE. NREs share sequence similarity with FBEs:
both consist of a highly conserved UGUR (R represents A/G)
sequence followed by either one (for the NRE) or two (for the
FBE) less conserved nucleotides and then AU (Opperman
et al., 2005). The element within the egl-4 30UTR can be aligned
with the hunchback NRE (Zamore et al., 1997) in two registers as
well as the FBF-binding gld-1 FBE (Crittenden et al., 2002)
(Figure 1C; Opperman et al., 2005). To determine whether this
GST-tagged protein in a gel mobility shift assay. Both recombi-
nant Drosophila Pumilio (GST-PUM-HD) (Zamore et al., 1997)
and C. elegans FBF-1 bound and shifted the electrophoretic
mobility of a synthetic 30 nucleotide RNA oligomer containing
the wild-type egl-4 element (Figure 1D, left gels). The affinity of
Drosophila Pumilio for the egl-4 element (Kd = 43 nM) was
comparable to its reported affinity for the canonical NRE
(Kd = 25nM) (Zamore et al., 1997). Likewise, the affinity of FBF-1
for the egl-4 NRE element (Kd = 75 nM) was also close to its re-
ported affinity for the gld-1 FBF (Kd = 23 nM) (Bernstein et al.,
2005). Neither protein bound appreciably to the corresponding
sequence from the mutant ky95 allele (Figure 1D, right gels). As
the egl-4 element bound either the Drosophila Pumilio or the
to as an NR/FBE.
The PUF FBF-1 Is Required in AWC for Adaptation
to Butanone and Isoamyl Alcohol
To determine whether FBF-1 is expressed in the same neuron
that EGL-4 acts to promote adaptation (L’Etoile et al., 2002),
we placed the GFP reporter under the control of sequences
2 Kb upstream of the start site for FBF-1 transcription. GFP
was expressed in both AWC neurons (Figure 2A), suggesting
that FBF-1 is normally expressed in the same neurons as its
proposed target, EGL-4.
We then used our adaptation paradigm (Figure 1A) (Colbert
and Bargmann, 1995) to determine whether FBF-1 is required
for adaptation to AWC-sensed odors. The fbf-1(ok91) null strain
was proficient at sensing the odors benzaldehyde, butanone,
and isoamyl alcohol (Figures 2B and S1), but failed to adapt to
butanone and was compromised for adaptation to isoamyl
alcohol (Figure 2B). This defect was somewhat odor specific
since it was capable of benzaldehyde adaptation (Figure 2B).
Given that EGL-4 acts cell-autonomously within the AWCs to
promote adaptation (L’Etoile et al., 2002), we asked if FBF-1
does the same. When fbf-1 mutants expressed FBF-1 solely in
the pair of AWCs and one other neuron pair (AWBs), they were
able to adapt to butanone as well as wild-type (Figure 2C).
Thus, FBF-1 is likely to act in the same neurons as its proposed
translational target, EGL-4 (L’Etoile et al., 2002).
FBF-1 Is Required during Odor Exposure for Adaptation
PUF proteins are required for a variety of developmental deci-
sions (Wickens et al., 2002). To determine if FBF-1-mediated
translational control is required for development of the AWC
neuron or during odor exposure, we placed the fbf-1 cDNA
under the control of the heat-shock promoter (Stringham et al.,
1992) and expressed this construct in the fbf-1(ok91) strain.
When the resulting strain was exposed to heat 2 hr prior to
odor exposure, it adapted as well as wild-type (Figure 2D, dark
gray). Heat had no effect on the behavior of the parental fbf-1
(ok91) strain (Figure 2D, medium gray). This indicates that
FBF-1 is required in the adult neuron, at the time of odor expo-
sure, for adaptation. Furthermore, AWC neuronal cell fate, as as-
sessed by str-2 promoter expression (Troemel et al., 1999), was
not altered in fbf-1-defective animals (Table S1).
The Nanos Protein NOS-1 and the Poly(A) Polymerase
GLD-3 Are Required for Adaptation to a Wide Range
To determine whether proteins that interact with PUFs are
tion in mutants that lack NANOS. nanos acts with pumilio to
repress the Drosophila NRE-containing hunchback message
(Sonoda and Wharton, 1999). Three nanos-related genes in
C. elegans play partially redundant roles in gonad development
(Kraemer et al., 1999). Of these, NOS-1 was required for wild-
type adaptation to all AWC-sensed odors tested (Figure 3A).
NOS-3, however, was not required for adaptation to any odor
tested (Figure 3A). Thus, though both PUF and NOS proteins
were both required to promote adaptation, the nos-1 mutants
were defective for adaptation to a wider range of odors than
the fbf-1(ok91) null mutants.
Further genetic evidence that translational regulation is
required for adaptation came from our observations of mutants
that lack the cytoplasmic poly(A) polymerase GLD-3 (Eckmann
et al., 2002; Wang et al., 2002). The poly(A) polymerase GLD-2/3
has been proposed to relieve PUF-mediated repression by
increasing poly(A) tail length, thereby increasing message
stability and/or translation (Eckmann et al., 2002; Suh et al.,
2006). We found that the poly(A) polymerase subunit-defective
gld-3(ok308) strain failed to adapt to any AWC-sensed odor we
lation and/or RNA stability. Since the egl-4 30UTR does not
contain a cytoplasmic poly-Adenylation element (CPE), egl-4
nents of the adaptation machinery may be targeted by GLD-2/3.
Genetic Evidence that the egl-4 NR/FBE Positively
The NR/FBE-mutated egl-4(ky95), the fbf-1 null, the nos-1 null
and the gld-3 null strains all fail to adapt to prolonged odor
exposure. Thus, they may all work in the same direction: to allow
EGL-4 to promote adaptation. Since the n479 loss-of-function
A PUF Activates Translation and Sensory Adaptation
60 Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc.
allele of egl-4 is defective for adaptation (L’Etoile et al., 2002),
this suggests that mutations in either the fbf-1 pathway or the
NR/FBE decrease levels of EGL-4 relative to wild-type. By
contrast, previous reports showed that disruption of either puf
genes or their targets actually increased the targeted gene’s
expression (Gu et al., 2004; Crittenden et al., 2002).
Cell-Biological Evidence that PUF Binding May Direct
the Subcellular Localization of EGL-4 Translation
To determine if the NR/FBE control element and FBF-1 direct where
EGL-4 is translated within the AWC of a living animal, we utilized the
photoconvertable stony coral protein Kaede (Ando et al., 2002) as
a reporter for new protein synthesis. Kaede fluorescence is
Figure 2. FBF-1 Is Required in the AWC Sensory Neuron at the Time of Odor Exposure to Promote Adaptation to Butanone
(A) FBF-1 is expressed in the AWC neurons. Fluorescence micrograph of a worm expressing GFP under the control of 2 kB of the fbf-1 promoter region (pfbf-1).
Fluorescence was observed in both AWC neurons (marked with an arrowhead), both AWB neurons (not marked), and one unidentified head neuron (not in this
plane of focus).
(B) While fbf-1(ok91) null mutant worms were unable to adapt to butanone (***p < 0.0001, n = 36), and were defective for adaptation to isoamyl alcohol
(**p = 0.0024, n = 19), they were fully capable of adapting to benzaldehyde (p = 0.1758, n = 13).
(C) Full-length FBF-1 expressed in AWC and AWB under the odr-1 promoter allowed fbf-1(ok91) mutants to adapt to butanone like wild-type (p = 0.6006, n = 4).
This was significantly different from the starting fbf-1(ok91) strain (**p = 0.0075, n = 4).
under the control of the heat-shock promoter were as defective for adaptation as the fbf-1 null worms alone (compare third and fifth pair of bars). When the
transgenic strain were exposed to ?30?C for 60 min, 2 hr prior to odor exposure, they adapted like wild-type animals (compare second and sixth pair of bars).
This adaptation was significantly different from the same strain before heat shock (**p = 0.0084, n = 7) and the heat-shocked fbf-1(ok91) (*p = 0.0264, n = 7). All
assays were performed with >50 animals per assay on separate days. p values were obtained using a two-tailed test. Error bars denote SEM.
A PUF Activates Translation and Sensory Adaptation
Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc. 61
et al., 2002). After photoconversion of Kaede to red, only newly
synthesized protein will fluoresce green. Kaede coding sequences
were placed upstream of either the wild-type or ky95-mutated egl-4
synthesized Kaede, a myristoylation signal (Adler et al., 2006) was
added to it. Myristoylation directs the newly synthesized protein to
the nearest plasma membrane thus increasing our chance of deter-
mining the site of new protein synthesis. Since the promoter, 50UTR
of the reporter protein should only result from differences in mRNA
processing and trafficking. These reporters were expressed from
transgenic arrays in either wild-type or fbf-1(ok91) mutant animals.
by quantitative real time PCR (Figure S2). Furthermore, the wild-type
reporter was examined in populations of siblings that contained the
same array but had either wild-type or fbf-1(ok91) genotype.
In the wild-type animal, newly synthesized Kaede under the
control of the wild-type egl-4 30UTR was observed in the sensory
Figure 3. The NANOS-Related Molecule
NOS-1 and the Poly(A) Polymerase Subunit,
to AWC-Sensed Odors
(A) NOS-1 deficient, nos-1 (gv5) worms failed to
adapt to the AWC sensed odors: benzaldehyde
(***p = 0.0002, n = 9), butanone (***p < 0.0001,
n = 9), and isoamyl alcohol (***p < 0.0001 n = 12).
In contrast, NOS-3-defective
(q650)] adapted to all three odors.
(B) Animals that lack the poly (A) polymerase
subunit gld-3(ok308) were unable to adapt to the
AWC-sensed odors: benzaldehyde (*p = 0.0117,
n = 5), butanone (*p = 0.0127, n = 6), and isoamyl
alcohol (***p = 0.0007, n = 8). Assays were per-
formed on separate days with >50 animals per
assay. p values were obtained using a two-tailed
test. Error bars denote SEM.
cilia as well as throughout the AWC
neuron (Figures 4A and 4B). When we
compared this to the subcellular distribu-
tion of newly synthesized Kaede under
the control of the ky95 mutant 30UTR,
we found that accumulation in the cilia
and in the cell body was 4.6- and 3.6-
fold lower respectively while accumula-
tion in either dendrites or axons was not
left to right panels; Figure 4B, compare
light to dark gray bars). Likewise, when
we compared expression from the wild-
type 30UTR reporter in wild-type animals
with its expression in sibling-derived
fbf-1(ok91) mutant animals, we found
that accumulation of newly synthesized
Kaede was significantly lower within the
cilia and cell body of the mutants (2.7-
and 3.9-fold lower, respectively) while
accumulation in the axons and dendrites was not significantly
different (Figure 4B). These results, however, have enough
statistical variation to warrant caution. The fbf-1 mutant back-
ground could possibly affect protein transport but it is unlikely
that the change in the 30UTR of the reporter would affect protein
trafficking. Thus, new synthesis of Kaede in the cell body and
cilia probably requires an intact NR/FBE and FBF-1.
Cell-Biological Evidence that FBF-1 Enhances
When we summed expression of the Kaede reporter throughout
the cell, we found that the NR/FBE mutations decreased overall
reporter expression appreciably. In wild-type animals, Kaede
recovery was 2.8-fold higher when the reporter contained the
wild-type NR/FBE than when it contained the mutant ky95
sequences (Figure 4C). Likewise, loss of the FBF-1 protein
reduced translation of the wild-type reporter to the levels seen
with the NR/FBE mutant reporter (2.5-fold) (Figure 4C). This indi-
A PUF Activates Translation and Sensory Adaptation
62 Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc.
tory adaptation. This positive regulation of translation by a PUF
and its target-binding site is in contrast to previous reports in
The increased translation we observed could be explained if
FBF-1 increases either translation rates or message levels. We
asked whether the loss of FBF-1 decreased our reporter’s
message levels. Using quantitative real-time reverse transcrip-
tase-mediated PCR (qRTPCR), we found that the ratio between
the reporter’s message level in wild-type as compared to fbf-1
(null) mutant animals was ?1.1 (Figure 4D). Thus, the message
levels are not appreciably changed by the loss of FBF-1 and
the FBF-1-dependent increase in reporter expression is likely
a function of an increase in translation rather than in message
The NR/FBE Mediates Odor-Induced
To determine if egl-4’s 30UTR provides dynamic odor-induced
regulation of EGL-4 translation, we developed a luciferase
reporter that would allow us to monitor rapid changes in expres-
sion. We placed a destabilized luciferase (30 min half-life) (Prom-
ega) under the control of an AWCON-specific promoter (pstr-2)
and flanked it with the egl-4 50and 30UTRs. To assess the role
of the 30UTR in odor-responsiveness, we altered this reporter
by replacing the egl-4 30UTR with the 30UTR of the muscle cell-
specific myosin unc-54 gene. To elucidate the role of the NR/
FBE and its flanking sequences in this process, we mutated
the egl-4 30UTR to mimic the ky95 form or deleted key regulatory
elements from this sequence (NR/FBE delete) (Figure 5A). Lucif-
erase values were normalized to numbers of copies of luciferase
genes in the population sampled (for expression from each line
see Supplemental Data). A population of animals was exposed
to odor for ?1 hr. Subsequently, one portion of the population
was subjected to a chemotaxis assay while the other portion
was subjected to a luciferase assay. Importantly, expression of
the transgenes did not alter behavior (Figure 5B).
Since EGL-4 is required for both odor sensation (Daniels
et al., 2000) and rapid short-term adaptation (L’Etoile, et al.,
2002), we expected that naive populations should express
the reporter at relatively high levels. If robust long-term adapta-
tion requires additional EGL-4 expression, we hypothesized
the naive levels that the increase would correlate with the
strength of adaptation. That is, as the animals become less
responsive to odor, they might also increase EGL-4 and lucif-
erase expression. When we examined populations that ex-
pressed luciferase under the control of the wild-type egl-4
inversely with the CI only after animals had been exposed to
odor (Figure 5C, bottom). This correlation was not observed
in the naive populations (Figure 5C, top). Thus, a decrease in-
chemosensation is strongly correlated with increased expres-
sion from the egl-4 30UTR and this relationship is not seen
unless the animals have been exposed to odor for a prolonged
To determine whether the correlation between luciferase
expression and behavior was dependent upon the egl-4 30
UTR, we examined animals in which luciferase was under the
control of the unc-54 30UTR. Though the behavior of these
animals was indistinguishable from those carrying the egl-4
30UTR (Figure 5B), there was no correlation between CI and
luciferase values either in naive or odor-exposed populations
(Figure 5C). Thus, the 30UTR itself seems to be the target of
regulation by prolonged odor exposure. We next asked whether
the NR/FBE might be required for the odor responsiveness of
the egl-4 30UTR element. Indeed, changing it to mimic the
ky95 30UTR or deleting it entirely abolished the odor-induced
(Figure 5C). Thus, correlation between behavior and egl-4
30UTR reporter expression depends upon an intact NR/FBE.
This correlation may be important for long-term adaptation
since the egl-4(ky95) mutant strain is unable to adapt to pro-
longed exposures of odor (L’Etoile et al., 2002). Further, since
FBF-1 binding is diminished by the ky95 mutation, this indicates
that the odor responsiveness of the 30UTR depends upon PUF
binding to this element.
Since the strength of adaptation can be measured by the
decrease in chemotaxis index (CI) and the longer the odor
(Colbert and Bargmann, 1995; L’Etoile et al., 2002), we decided
to compare luciferase reporter expression in populations with
high and lowCIs. When populations of naiveanimals expressing
the luciferase reporter under the control of the wild-type egl-4
30UTR that chemotaxed well (CI > 0.8) were compared to iden-
tical animals that had adapted robustly (CI < 0.2), we saw that
luciferase was 2-fold higher in the adapted populations than in
the naive populations (Figure 5D). This odor regulation was
dependent upon the 30UTR as we failed to see an odor-
dependent increase in luciferase levels when the egl-4 30UTR
was replaced with the unc-54 30UTR or when the NR/FBE was
deleted from our reporter (Figure 5D). Thus, though EGL-4 is
required in the naive animal, prolonged odor-exposure acts
via the NR/FBE element within the egl-4 30UTR to increase
EGL-4 expression. This indicates that FBF-1 promotes adapta-
tion by enhancing EGL-4 translation.
The PUF-Binding Site and Its Adjacent Sequences
Enhance Basal Expression
Since both activated and basal levels of expression require PUF
control, we wanted to understand how FBF-1 binding to the NR/
FBE might affect basal expression of egl-4. We examined lucif-
erase expression in naive animals removed directly from food.
Basal levels of expression required FBF-1: expression from the
wild-type reporter was ?2.4-fold lower in fbf-1(ok91) mutant
animals as compared to wild-types and loss of FBF-1 binding
to the egl-4(ky95) 30UTR resulted in ?4.5-fold lower expression
(Figure 5E). Since expression from the egl-4(ky95) 30UTR
reporter was roughly the same in either fbf-1(ok91) or wild-type
animals (Figure 5E, compare third and fourth bars) it is unlikely
that FBF-1 is acting through additional targets to promote
To understand how the NR/FBE and its surrounding
sequences might contribute to basal EGL-4 expression, we en-
gineered specific changes in the 30UTR (Figure 5A). Selective
deletion of the NR/FBE or wholesale replacement of the 30UTR
A PUF Activates Translation and Sensory Adaptation
Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc. 63
Figure 4. The egl-4 NR/FBE Promotes Translation within the AWC Neuron
(A) (Top)Cartoonsof myristoylated Kaede expression reporters under the control of the str-2promoter wereflanked withthe egl-4 50UTR and either the wild-type
egl-4 30UTR or the egl-4(ky95) 30UTR. These were expressed in either wild-type (left and right columns of images) or fbf-1(ok91) animals (middle column).
pstr-2::RFP (red) delimits the AWC neuron (top and bottom rows of images). The parts of the AWC neuron are marked with arrows (upper left). The top row of
panels: red and green channels immediately after photoconversion. The second row panels: residual unconverted Kaede in the green channel immediately after
photoconversion. The third row of panels: the green channel reveals the subcellular location of accumulated Kaede of the same animal after 3 hr of recovery on
food. The fourth row of panels: the merged red and green channels of the same animal.
(B) Kaede synthesis near the sensory cilia and cell body depends on the 30UTR and FBF-1. Newly synthesized Kaede under the control of the wild-type 30UTR
expressed in the fbf-1(ok91) mutant animals was 2.7-fold lower in the cilia (*p = 0.0233, n = 5) and 3.9-fold lower in the cell body (*p = 0.028) than when the same
reporter transgeneis expressedinwild-typeanimals.Inwild-typeanimals expressingKaedeunderthecontrol oftheegl-4(ky95)30UTR,newlysynthesizedKaede
controlof the wild-type 30UTR. Inthe axon and dendrite, the Kaede expression wasnot significantlydifferent inanimals that bear the ky95mutation (p = 0.25) or in
animals that lack fbf-1 (p = 0.17). The copy number of the wild-type and ky95 30UTR reporter was the same in each line (Supplemental Data and Figure S2).
p values were obtained using a two-tailed unpaired t test.
(C) AWC-wide Kaede expression was determined by integrating Kaede levels over the whole cell using the images analyzed in (B). Over-all Kaede levels were
2.6-fold higher in wild-type worms expressing Kaede flanked with the wild-type egl-4 30UTR than in fbf-1(ok91) mutants expressing the same transgene
A PUF Activates Translation and Sensory Adaptation
64 Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc.
with that of unc-54 30resulted in 2-fold lower expression
(Figure 5F). This lower expression was not changed by replace-
ment of the NR/FBE with the gld-1 FBEa (Figure 5F, second and
third bars) indicating that a PUF-binding site per se may not be
sufficient to either promote or repress basal translation.
However, FBF-1 may not interact with the hybrid UTR at all since
its expression was not affected in the fbf-1 mutant strain
(Figure S4). In fact, residues both 50and 30of the FBEa affected
Pumilio binding in vitro (Bernstein et al., 2005). Thus, the context
of the PUF-binding site may be critical for the interaction and we
this, wewere not able to ‘‘reprogram’’ the unc-54 30UTR by addi-
tion of either the NR/FBE or FBEa (Figure S5).
To assess how sequences flanking the NR/FBE might affect
PUF-mediated basal translation, we deleted 4 additional bases
30of the original NR/FBE deletion (NR/FBE delete+4). The addi-
tional 4 base deletion is predicted to abolish the binding of the
microRNA mir-84 (the predicted change in free energy upon
binding goes from ?18.27 kCal/mol to no predicted binding
using Miranda; Enright et al., 2003) and to decrease the binding
of the microRNA let-7(free energy of binding goes from ?18.27
Kcal/mol to ?14.45 kCal/mol). The removal of these residues
decreased expression further (compare Figure 5F, second and
fourth bars), indicating that flanking sequences are crucial for
basal translation. Since the Miranda (Enright et al., 2003)
predicted seedsequence for
if this site might contribute to basal reporter expression. We
found that selective deletion of the seed sequence (Figure 5A)
decreased expression by ?5.2-fold (Figure 5F). Originally, we
had hypothesized that microRNA might repress translation by
binding to these sites and that FBF-1 might displace the micro-
RNAs and thereby activate translation. Since high levels of basal
reporter expression require the predicted microRNA seed
sequence,it isformally possible thatmicroRNAs positively affect
cells but thus far, has not been reported in a living, behaving
This work demonstrates the substantial role that a 30UTR
element can play in determining the behavior of an entire
organism. We show that an NRE-containing sequence (the NR/
FBE) lying within the 30UTR of the key adaptation-promoting
factor, egl-4 is bound by FBF-1 and regulates EGL-4 expression
in adapted animals. This element may also help localize EGL-4
translation near the AWC sensory cilia. To gain mechanistic
insight into how the egl-4 30UTR enhances expression, we
examined both the NR/FBE and its flanking sequences. We
found that the NR/FBE itself is required for high levels of reporter
expression in naive worms. In long-term adapted worms, odor
increases expression over naive levels in an NR/FBE-dependent
fashion. We provide both genetic and cell biological evidence
that in an adult neuron, the PUFs that repress translation in other
cells may promote plasticity by allowing odor stimulation to acti-
vate translation and this may occur near the cell body and
sensory cilia. These results indicate that 30UTR elements can
act as stimulus-responsive translational enhancers in a sensory
neuron. Finally, this PUF-dependent increase in translation is not
accompanied by an appreciable increase in message levels,
indicating that instead of stabilizing transcripts, FBF-1 is likely
to directly activate translation.
Our observation that a PUF and its target site can activate
translation is at odds with the way these factors have been
shown to work in C. elegans development (Wickens et al.,
2002), in the fly motor neurons (Mee et al., 2004), at the fly neuro-
muscular junction (Menon et al., 2004), or in yeast mating type
switching (Gu et al., 2004) where they have been shown to
repress translation. However, in meiotic maturation of Xenopus
oocytes, PUF binding sites were recently shown to work in
conjunction with a cytoplasmic polyAdenylation element (CPE)
within the cyclin B1 30UTR to upregulate translation (Pique ´
et al., 2008). Since the egl-4 30UTR does not contain an identifi-
able CPE, the FBF-1-binding site may be working in a distinct
manner to induce translational activation perhaps with the aid
30UTR-Mediated Regulation of Translation and Behavior
How does a sensory neuron assess the duration or repetition of
stimulation in order to alter its excitability? Our studies indicate
that prolonged odor exposure regulates new protein synthesis
of the EGL-4 kinase via its NR/FBE. This 30UTR-based strategy
is different from the protein translocation and trafficking based
strategy used by visual sensory neurons (Calvert et al., 2006).
What we see in the C. elegans AWC olfactory sensory neuron
may be more akin to what happens in interneurons within
ticity promoting proteins. For example, odor association in flies
and long-term memory formation in mice both require regions
of the CamKIIa 30UTR to restrict expression of the kinase
spatially and temporally (Miller et al., 2002; Ashraf et al., 2006).
Furthermore, long-term facilitation in Aplysia requires highly
localized new protein synthesis specifically at the stimulated
synapse (Martin, 2002; Martin et al., 1997). How is translation
directed to specific regions within the neuron? Mechanisms
that limit translation both spatially and temporally have emerged
for CamKII: both dendritic transport and activity-dependent
degradation of the RISC complex in flies and the cytoplasmic
poly adenylation element binding proteins (CPEBs) in mice and
(*p = 0.0165, n = 6). Kaede recovery was 2.8-fold higher in wild-type worms expressing Kaede flanked with the wild-type egl-4 30UTR than in wild-type worms
expressing Kaede with the ky95 30UTR (*p = 0.0443, n = 6). Newly synthesized Kaede was quantified from confocal images using Volocity software (see Supple-
(D) Kaede mRNA levels are the same in both wild-type and fbf-1(ok91) mutant worms. Kaede mRNA levels did not differ significantly between these strains
(p = 0.416). Real-time PCR was performed on cDNA made from worms of each genotype. Units of Kaede mRNA/unit of HMG CoA mRNA were determined
for each and the ratio of this value is shown.
All error bars denote SEM.
A PUF Activates Translation and Sensory Adaptation
Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc. 65
Figure 5. The egl-4 NR/FBE and a Predicted let-7/mir-32/84/265 miRNA Seed Sequence Both Promote Expression of a Luciferase-Based
Reporter in the AWC Neuron
(A) Diagram of Luciferase-based reporters. All were placed under the control of the str-2 promoter and each contains the indicated 30UTR. Upper brackets
indicate the nucleotides that were deleted. The longer NR/FBE delete in Figure 5F extends 4 n.t. 30of the NR/FBE brackets.
(B) Chemotaxis assays were performed on naive and odor-exposed animals carrying the indicated transgenes.
(C) (Top) The chemotaxis behavior (CI) of naive animals (x axis) to butanone was not correlated with luciferase expression (y axis) in any of the reporter strains
examined. When the wild-type egl-4 30UTR flanks this reporter, Pearson’s r is ?0.12, p = 0.74; for the unc-54 30UTR, Pearson’s r is –0.71, p = 0.11; for the
ky95 30UTR, Pearson’s r is 0.04, p = 0.96; and for NR/FBE delete, Pearson’s r is 0.17, p = 0.78. (Bottom) The chemotaxis index of odor-exposed animals
correlated well with luciferase expression when the wild-type egl-4 30UTR flanks this reporter (one independent line) (Pearson correlation r = ?0.7324,
*p = 0.016, and Spearman’s correlation r = ?0.7939 and **p = 0.0088). Expression was uncorrelated with behavior after odor-exposure when the egl-4
30UTR (one line) was replaced by the unc-54 30UTR (Pearson’s r = 0.01, p = 0.98), the NR/FBE was mutated to mimic the ky95 allele (two lines) (Pearson’s
r = ?0.74, p = 0.26), or the NR/FBE was deleted (one line) (Pearson’s r = ?0.33, p = 0.58). Each point represents a single assay in which a population
was divided and a portion was subjected to a chemotaxis assay and the other portion to luminometry followed by qRT-PCR to obtain units of luciferase
per unit of transgenic DNA.
A PUF Activates Translation and Sensory Adaptation
66 Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc.
Aplysia limit its translation to specific spatiotemporal windows
(see Sutton and Schuman, 2006, for review). Our results indicate
that a PUF’s interaction with its target may also be able to
localize translation within the neuron, whether this localized
translation is also increased in response to an odor stimulus
remains to be seen.
Both the AWC neuron and the interneurons within a circuit
respond to multiple stimuli: each AWC neuron probably senses
tens of odors (Troemel, 1999). Cells that respond to multiple
stimuli might require spatial localization of key plasticity
promoting factors; translational control over protein synthesis
may provide this precision. PUF-regulated translational control
over EGL-4 expression could provide a measure of odor speci-
ficity to the adaptation process by spatially localizing translation.
Alternatively, since we have shown that both classes of PUFs
bind to the egl-4 30UTR and many of these are expressed in
AWC (data not shown), unique combinations of PUFs could
interactwith theNR/FBE in a way thatwould allow for adaptation
to distinct odors. Thus, each PUF could provide a unique contri-
bution to odor adaptation. Alternatively, combinations of PUFs
could regulate levels of other key adaptation-promoting factors.
The fact that loss of one of the 11 C. elegans PUF’s, FBF-1,
affects fewer odors than loss of NOS-1 or GLD-3, the other
members of the PUF translational control pathway, suggests
that other PUFs might interact with NOS-1 and GLD-3 to
promote adaptation to the additional AWC-sensed odors
perhaps via other targets.
Translational Activation Is Directed
by the 30UTR Sequence
This work provides an important insight into translation: a 30UTR
element can allow a specific environmental stimulus to dynami-
cally activate translation. This may require an adjacent predicted
microRNA-binding site. A similar scenario was observed for the
repression of the hbl-1 30UTR transcript: both PUF-9 and the
microRNA let-7 were required for proper timing and full repres-
sion of the hunchbacklike transcript (Nolde et al., 2007). Thus,
there is precedent for Pumilios and microRNAs collaborating to
regulate translation. In fact, recent bioinformatic evidence has
indicated that PUF and microRNA-binding sites are adjacent to
each other more frequently than would be predicted if their
placement was random, suggesting that these two translational
control elements may act together to regulate gene expression
(Galgano et al., 2008).
Our finding that an otherwise repressive 30UTR element acti-
vates translation is reminiscent of reports on the regulation of
tumor necrosis factor a (TNFa) (Vasudevan and Steitz, 2007).
The TNF a 30UTR contains an AU-rich element (ARE) that can
either repress or activate translation depending upon the cellular
milieu rather than the ARE sequence itself (Vasudevan et al.,
2007). In the postmitotic adult neuron a similar scenario may
play out: odor-induced changes in the cellular cohort of PUF-
associated proteins and/or RNAs may mediate translational
activation. Alternatively, the NR/FBE sequence itself may direct
activation. Thus, it will be interesting to determine if the novel
NR/FBE element found within other 30UTRs also activates
message translation. Whether they also activate or repress
translation, independent sequence elements within the egl-4
30UTR such as the PUF and micro-RNA binding sites could
generate a ‘‘code’’ for behavior that would enable specific
aspects of a behavior to evolve independently as each element
is retained or lost.
Finally, in mice, the PUF PUM2 (Xu et al., 2007), NANOS1
(Haraguchi et al., 2003), and the mammalian GLD2 poly(A)
polymerase (Rouhana etal.,2005)areall expressed inthe hippo-
campus suggesting a role for this cohort of proteins in memory
formation. It will be interesting to see whether mice that lack
these proteins exhibit any behavioral phenotypes and whether
the PUF pathway activates translation in the context of mamma-
Strains used in this work include N2 as wild-type; JK3022 fbf-1(ok91) (9X out
crossed); JH1270 nos-1(qv5) (6X outcrossed); JK2589 nos-3 (q650 (8X out-
crossed)); BS3493 gld-3(ok308)/mIn1[dpy-10(e128) mIs14] II (3X outcrossed).
Nematode strains used in this work were provided by the Caenorhabditis
Genetics Center, which is funded by the NIH National Center for Research
Full-length cDNA encoding FBF-1 (yk322e3 from Yuji Kohara) was PCR ampli-
fiedandfusedtoGSTsequencesinpGex3T usingtheBamH1and EcoR1sites
incubated with 100 pM32P-labeled wild-type or ky95 30-mer RNA oligomers
(Dharmacon) (Bernstein et al., 2005). Gels were resolved for 2 hr, dried, and
quantified using Image Quant and Kds were estimated by nonlinear regression
curve fitting by GraphPad Prism software. GST-tagged Pum HD (generous gift
from Phillip Zamore) was purified as previously described (Zamore et al., 1997)
(D) Luciferase values in populations of naive worms that chemotaxed well (CI > 0.8, light gray) were compared to those that adapted robustly (CI < 0.2, dark
gray bars). Luciferase expression was 1.85-fold higher in animals that adapted well than in the naive animals (p = 0.05, n = 3). Error bars denote the SEM.
(E) Luciferase reporter expression in the AWCONneuron in naive animals was examined as a function of the 30UTR. The wild-type egl-4 30UTR (five indepen-
dent lines, see Supplemental Data on details of expression from each line) in wild-type animals conferred 2.4-fold more expression (n = 40) than when it was
expressed in fbf-1(ok91) mutant animals (three lines, p = 0.0061, n = 15), 4.5-fold higher than the ky95 30UTR expressed in wild-type animals (two lines,
p = 0.0003, n = 15) and 6.2-fold higher than the ky95 30UTR expressed in fbf-1(ok91) (two lines p < 0.0001, n = 11). fbf-1(ok91) animals expressing the
wild-type 30UTR showed 2.3-fold more luciferase expression than fbf-1(ok91) animals expressing the ky95 30UTR (p = 0.0025). Expression from the ky95
30UTR reporter was not significantly different in wild-type and fbf-1(ok91l) animals (p = 0.1591).
(F) The NR/FBE and flanking sequence elements promote basal levels of expression from the egl-4 30UTR. In wild-type worms, the egl-4 30UTR (five lines) was
expressed 2.3-fold more than the NR/FBE deleted 30UTR (one line, **p = 0.0077, n = 9), 2.8-fold more the gld-1 FBEa substitution (three lines, **p = 0.0028,
n = 25), 4.8-fold more than the NR/FBE + 4 bp delete (three lines, **p = 0.0002, n = 9) and 5.2-fold more than the let-7/mir-32/84/265 deletion (two lines,
***p = 0.0002, n = 8). The unc-54 30UTR drove 2.4-fold less expression than the WT egl-4 30UTR one line, p = 0.0449, n = 8). Luciferase/unit DNA values for
wild-type egl-4 30UTR, gld-1-subsitiuted FBE 30UTR and unc-54 30UTR lines did not follow a normal distribution; thus, p values comparing means were
obtained using a Welch-corrected two-tailed t test using Prism software. Error bars denote SEM.
A PUF Activates Translation and Sensory Adaptation
Neuron 61, 57–70, January 15, 2009 ª2009 Elsevier Inc. 67
and see Supplemental Data for further details. Binding reactions were
performed as described (Bernstein et al., 2005) for FBF-1.
All strains were grown as described (L’Etoile et al., 2002) except gld-3 which
was grown at 20?C and shifted to 25?C for 4 hr before assaying. Odors were
used at the following concentrations: 7 ml of benzaldehyde per 100 ml Sbasal,
10–12 ml of butanone per 100 ml S basal (L’Etoile et al., 2002). Isoamyl alcohol
adaptation was performed as described (Colbert and Bargmann, 1995). Each
chemotaxis assay was performed with a minimum of 50 animals. Statistical
tests were performed by GraphPad Prism software.
The full-length cDNA encoding FBF-1 was placed under the control of
podr-1::RFP. See Supplemental Data for details of this construct and genera-
tion of the transgenic lines.
Transgenic worms expressing either the wild-type or ky95 egl-4 30UTR down-
stream of luciferase (see SupplementalData for the details of the cloning) were
immobilized on 2% agarose pads with 1 mM NaN3. Kaede was photocon-
verted by irradiating with 365 nM UV light for 45 s using the 633 objective.
The irradiated worms were then imaged in the green and red fluorescence
channels using a 488 and 543 nM scanning laser. Worms were recovered
from pads with S-Basal and allowed to recover on seeded plates for 3 hr prior
to reimaging. Worms were imaged on a Zeiss LSM 510 META laser-scanning
confocal microscope and images were quantified using Volocity software
various permutations, see Supplemental Data for details of the generation of
these constructs) were grown for 5 days on HB101 at 23?C–25?C. Worms
were collected by washing off the cultured plates with S-Basal and were
washed free of E. coli by washing two times in S-Basal and two times in water.
Between washes, animals were spun for 6 s in a nanofuge and the final spin
was at 12,000 RPM in an Eppendorf centrifuge 5415D for 1 min. Excess water
wasremovedandworms werefrozenimmediately inliquidnitrogenandstored
at ?80?C. Worms were thawed in a water bath at RT and DMSO was added to
a final concentration of 10%. Animals were vortexed on maximum for 10 s, re-
frozen in liquid N2, and thawed for 2 min in a RT water bath. This was repeated
four more times. Forty microliters of lysed worm suspension was used in
a Turner Biosystems 20/20 luminometer and total luminescence was
measured using the Promega Luciferase Assay System as described in
technical bulletin number 281. Immediately following luminometry, the sample
containing lysed worms and the luciferase cocktail was subjected to quan-
titative real-time PCR to determine transgene copy number (Supplemental
The Supplemental Data include Supplemental Experimental Procedures,
figures, and a table and can be found with this article online at http://www.
We would like to thank Shih-Yu Chen for his helpful insights; Justine Melo,
Damien O’Halloran, Jin Lee, Georgia Woods, Mark Lucanic, Christopher Sulli-
van, Bruce Draper, Brian Mulloney, Cori Bargmann, Maria Gallegos, Judith
Kimble, and our reviewers for critical reading of this manuscript and construc-
tive discussions; and Marv Wickens and Laura Opperman for corroborating
our findings and sharing unpublished data. We thank Yuji Kohara for cDNA
clones and Theresa Stiernagle and the CGC for strains; Pilai Sengupta for
pofm-1::GFP; Cori Bargmann and Miri Van Hoven for pstr-2::RFP; Peter
Lengyel, Ting Wen Cheng, and Danny Rozelle for the reporter constructs
and turning the egl-4 30UTR into a cassette; Philip Zamore for DPUM and
advice; Brad Hook for advice on gel shifts; and Laurel Beckett for statistical
counsel. We are also grateful to Mehrdad Matloubian and Amanda Kahn-Kirby
for help with QRT-PCR and comments on the manuscript; J. Michael Bishop
for use of his lab; and Robert Bailey for imaging and trouble-shooting; and
the W.M. Keck Program in Neuroscience Imaging. This work was supported
by the NIH Molecular Cellular Biology training grant T32GM007377-31 and
NIH T32DC008072 for J.A.K.; the Sandler Program in Biological Sciences
grant for A.G., and the Joe P. Tupin Intramural Grant Award program adminis-
tered by the Department of Psychiatry and Behavioral Sciences, UC Davis
School of Medicine Health Sciences Award and NSF 0317136 and NIH
R01DC5991 to N.D.L.
Accepted: November 5, 2008
Published: January 14, 2009
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