Specificity of ARGONAUTE7-miR390
Interaction and Dual Functionality
in TAS3 Trans-Acting siRNA Formation
Taiowa A. Montgomery,1,2Miya D. Howell,2,3Josh T. Cuperus,1,2Dawei Li,4Jesse E. Hansen,2Amanda L. Alexander,2
Elisabeth J. Chapman,1,2,5Noah Fahlgren,1,2Edwards Allen,2,3,6and James C. Carrington2,3,*
1Molecular and Cellular Biology Program
2Department of Botany and Plant Pathology
3Center for Genome Research and Biocomputing
Oregon State University, Corvallis, OR 97331, USA
4State Key Laboratory for Agro-Biotechnology, China Agricultural University, Beijing 100094, China
5Present address: Department of Biology, Indiana University, Bloomington, IN 47405, USA.
6Present address: Monsanto Company, Chesterfield, MO 63017, USA.
Trans-acting siRNA form through a refined RNAi
triggers entry of precursor transcripts into an RNA-
DEPENDENT RNA POLYMERASE6 pathway, and
sets the register for phased tasiRNA formation
by DICER-LIKE4. Here, we show that miR390-
ARGONAUTE7 complexes function in distinct cleav-
age or noncleavage modes at two target sites in
TAS3a transcripts. The AGO7 cleavage, but not the
noncleavage, function could be provided by AGO1,
the dominant miRNA-associated AGO, but only
when AGO1 was guided to a modified target site
through an alternate miRNA. AGO7 was highly selec-
tive for interaction with miR390, and miR390 in turn
was excluded from association with AGO1 due en-
tirely to an incompatible 50adenosine. Analysis of
AGO1, AGO2, and AGO7 revealed a potent 50nucle-
otide discrimination function for some, although not
all, ARGONAUTEs. miR390 and AGO7, therefore,
evolved as a highly specific miRNA guide/effector
protein pair to function at two distinct tasiRNA
miRNA and tasiRNA are distinct classes of small RNAs that guide
silencing of target RNAs through cleavage or nondegradative re-
pression mechanisms (Chapman and Carrington, 2007). miRNAs
arise from transcripts that adopt imperfect, self-complementary
foldbackstructures, whereastasiRNAs arise froma refinedadap-
miRNA-guided cleavage, which forms a discrete 50or 30end, and
then transcribed by RNA-DEPENDENT RNA POLYMERASE6
(RDR6). The resulting dsRNA is processed into siRNA duplexes
in end-dependent, 21 nucleotide steps by DICER-LIKE4 (DCL4)
(Allen et al., 2005; Gasciolli et al., 2005; Peragine et al., 2004;
Vazquez et al., 2004; Xie et al., 2005; Yoshikawa et al., 2005).
Effector complex formation involves strand separation and
selective association with an ARGONAUTE (AGO) protein.
Arabidopsis contains four characterized TAS gene families.
TAS1, TAS2, and TAS4 tasiRNA biogenesis initiates with
miR173- (TAS1 and TAS2) or miR828-guided (TAS4) cleavage
on the 50side of the tasiRNA-generating region, while TAS3
tasiRNAs form by miR390-guided cleavage on the 30side.
miR390 also interacts in a noncleavage mode with a second
site near the 50end (Axtell et al., 2006; Howell et al., 2007).
Features of the TAS3 family, including targeting by miR390, are
highly conserved in land plants (Allen et al., 2005; Axtell et al.,
2006, 2007; Talmor-Neiman et al., 2006). TAS3 tasiRNAs, but
not those from TAS1 or TAS2, are dependent on a specialized
ARGONAUTE, AGO7 (also called ZIP) (Adenot et al., 2006;
Fahlgren et al., 2006; Hunter et al., 2006). TAS3 tasiRNAs target
mRNAs encoding several AUXIN RESPONSE FACTORs (ARF3
and ARF4), negative regulation of which is necessary for proper
developmental timing and lateral organ development along the
adaxial-abaxial axis (Adenot et al., 2006; Fahlgren et al., 2006;
Garcia et al., 2006; Hunter et al., 2006).
The mechanisms for recognition and routing of transcripts
through the tasiRNA or RDR6/DCL4-dependent pathway are
not well understood. Axtell et al. (2006) proposed a two-hit
trigger mechanism, in which transcripts with two or more small
RNA target sites are preferentially routed into the RDR6/DCL4
pathway. This explains some aspects of TAS3 tasiRNA forma-
tion and the routing of several known, multiply targeted
transcripts (Axtell et al., 2006; Chen et al., 2007; Howell et al.,
2007), although not necessarily TAS1, TAS2, and TAS4 tasiRNA
biogenesis. How the factors associated with miR828, miR173,
and miR390 function to provide routing information remains an
unresolved problem. In this paper, we show that miR390 is
uniquely adapted to initiate TAS3 tasiRNA biogenesis due to
128 Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc.
its specific association with AGO7, and that AGO7-miR390
complexes function in two distinct modes to process and route
transcripts through the RDR6/DCL4 pathway. We also found
an AGO function that discriminates among small RNAs on the
basis of 50nucleotide identity.
Design and Validation of Syn-tasiRNAs
Based on the predictable phased pattern of tasiRNA formation
from the miR390-guided cleavage site, we generated TAS3a-
based synthetic (syn)-tasiRNAs to silence the Arabidopsis
PHYTOENE DESATURASE (PDS) transcript. Silencing of PDS
mRNA results in photobleaching of green tissues (Kumagai
et al., 1995). Canonical targeting rules were followed for syn-
tasiRNA development, including incorporation of a 50U and
perfect complementarity between syn-tasiRNA nucleotides 2–13
(from the 50end) and the mRNA target (Allen et al., 2005;
Jones-Rhoades and Bartel, 2004; Schwab et al., 2005). Two 35S
promoter-driven TAS3a-based constructs (35S:TAS3aPDS-1
and 35S:TAS3aPDS-2) with tandem syn-tasiRNAs in the 50
D7[+] and 50D8[+] positions (Allen et al., 2005) were developed
(Figure 1A). These positions normally yield tasiR2141 and
tasiR2142, which target ARF transcripts.
In Col-0 (wild-type, wt) plants expressing 35S:TAS3aPDS-1
and 35S:TAS3aPDS-2, photobleaching emanated from the
midrib and major veins and was more apparent from the adaxial
side (Figure 1B). PDS mRNA accumulation was suppressed by
33% in 35S:TAS3aPDS-1-transformed plants and 50% in
35S:TAS3aPDS-2-transformed plants, but was suppressed to
a slightly greater extent in midrib tissue compared to nonmidrib
leaf tissue (p < 0.05; Figures 1C and 1D). Cotyledons displayed
photobleaching that emanated from the apex along the petiole
(Figure 1B). Mild photobleaching was also detected in flowers
and siliques of 35S:TAS3aPDS-2-transformed plants. Syn-
tasiRNAs and the photobleaching phenotype were RDR6-,
DCL4-, and AGO7 (ZIP)-dependent (Figures 1B and 1E; Table
1). PDS mRNA levels were not significantly different between
Col-0 vector-transformed plants and rdr6-15, dcl4-2, and zip-1
35S:TAS3aPDS-2-transformed plants (p values between 0.26–
0.86, two-sample t tests; Figure 1C). These results are consistent
syn-tasiRNA system faithfully reflects the endogenous pathway.
Limited Activity and Expression of AGO7
Plants expressing 35S:TAS3aPDS-1 and 35S:TAS3aPDS-2
displayed strongest photobleaching in vasculature-proximal
tissue. To determine if expression patterns of known TAS3-
specific factors explain this pattern, promoter-GUS fusion
constructs were developed with TAS3a, MIR390a, MIR390b,
and AGO7 promoters. The GUS activity patterns in seedlings
expressing TAS3a:GUS, MIR390a:GUS, and MIR390b:GUS
(35S:GUS). In contrast, seedlings expressing AGO7:GUS had
activitythat was detected
(Figure 1F). Syn-tasiRNA formation was tested in the presence
or absence of ectopic AGO7 in N. benthamiana leaves. When
transiently coexpressed with 35S:MIR390a or 35S:MIR390b,
primarily inthe vasculature
the 35S:TAS3aPDS-2 construct failed to yield syn-tasiRNA
(Figure 1H; lanes 7 and 8). But when coexpressed with
35S:MIR390a or 35S:MIR390b and a 35S-promoter driven
AGO7 construct (35S:HA-AGO7; Figure 1G), 35S:TAS3aPDS-2
yielded relatively high levels of syn-tasiRNA (Figure 1H; lanes 9
and 10). The partial overlap between AGO7 promoter activity
and TAS3a-based syn-tasiRNA activity and the ability of
constitutively active AGO7 to overcome limitations to syn-
tasiRNA formation in N. benthamiana leaves suggest that
AGO7 is limiting in leaves.
Distinct Roles for miR390 at the TAS3a
50and 30Target Sites
TAS3 tasiRNAs originate from sequences between two miR390
target sites. In flowering plants, but not in moss or pine, the
miR390-target interaction at the 50proximal site contains key
mispairs that prevent cleavage, and conversion to a cleavable
miR390 target site inactivates the TAS3a locus (Axtell et al.,
2006). In contrast, the 30proximal site is cleaved across plant
species (Axtell et al., 2006). The unique roles of miR390 at the
two target sites were analyzed with single and combination
target site substitutions using 35S:TAS3aPDS-1 and 35S:
TAS3aPDS-2 in Col-0 and zip-1 plants. The 30site substitutions
generated miR171 or miR159 sites, or a nonrecognized site
(Figures 2A and S1A). The 50substitutions yielded an authentic,
cleavable miR171 site, or target sites for five miRNAs that
approximated the characteristics of the noncleavable miR390/
TAS3a 50target interaction, including mispairs or G:U pairs at
positions 9, 10, and 11 of the miRNA (Figures 2A and S2A; Table
S1). A construct with 50site substitutions that destroyed target-
ing was also generated (Figure S2A). A dual target site substitu-
tion construct contained cleavable versions of miR171 target
sites (Figure 2A).
The 30and 50target site substitution mutants were first tested
for transcript cleavage properties using a 50RACE assay with
transgenic plant extracts. Cleavage at the canonical position of
the authentically paired miR390, miR171, and miR159 30target
sites, but not at the destroyed target site (35S:TAS3aPDS-
30390mut), was detected in the majority of cloned 50RACE
products (Figures 2A and S1A). At miR171 target sites, cleavage
offset by three nucleotides from the canonical position was also
detected, likely a result of cleavage guided by the distinct
miR171b/c isoform (Figures 2A, S1A, and S1B). Cleavage was
not detected at the authentic 50miR390 target site from any
construct or at the destroyed 50site, and was rare at predicted
noncleavable heterologous 50target sites (Figures 2A and S2A)
(data not shown). Therefore, the 50and 30substitution variants
generally possessed target site cleavage properties as intended.
The miR171 and miR159 30target site substitution and paren-
tal constructs triggered photobleaching in 65%–97% of plants
(Figures 2A and S1A). Photobleaching was absent in plants
with a noncleavable 30target-site construct (35S:TAS3aPDS-
30390mut; Figure S1A). Syn-tasiRNA levels were not significantly
different in Col-0 plants with either the parental 35S:TAS3aPDS-
2 or the miR171 30target substitution construct (p value = 0.55,
two-sample t test), but importantly, photobleaching triggered
by both constructs was lost in zip-1 plants (Figure 2A). This
indicated that substitution of the 30miR390 target site with a
Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc. 129
130 Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc.
functional, alternative target site is tolerated, but does not over-
come the system requirement for AGO7.
In contrast to the functional 30substitutions, both cleavable
(35S:TAS3aPDS-50171cle-2 and 35S:TAS3aPDS-5030171cle-2)
and noncleavable (35S:TAS3aPDS-50171nc-2) miR171 target
substitutions at the 50site eliminated photobleaching and
reduced syn-tasiRNAs to background levels in Col-0 plants,
regardless of the identity of the 30target site (Figures 2A and
2B). Each of the other heterologous 50target sites, and the
destroyed 50site, resulted in low or nondetectable syn-tasiRNA
levels and loss of photobleaching (Figures S2A and S2B). It is
possible that loss of syn-tasiRNA was caused by misprocessing
of the transcripts, such that tasiRNAs were formed upstream (50)
of the 50target sites. However, we failed to detect small RNAs
using probes corresponding to sequences on the 50side of any
of the modified 50target sites (Figure S2B). The collective tar-
get-site substitution data indicate requirements for a 50miR390
tional 30cleavage site, though not necessarily a miR390 site.
Role of AGO7 in TAS3a Transcript Processing
To determine if AGO7 is required for cleavage of endogenous
TAS3a transcripts, 50RACE assays were done using wt and
mutant plants. 50RACE product corresponding to cleavage of
TAS3a transcript at the 30miR390 target site was detected in
Col-0 plants (Figure 3A). A slightly larger 50RACE product corre-
sponding to cleavage 33 bases upstream of the miR390 cleav-
age site was also detected (Figure 3A, lanes 1–3). As shown
previously (Allen et al., 2005), this product corresponds to cleav-
age guided by a secondary siRNA (TAS3a 50D2[?]) derived from
the TAS3a complementary strand. In rdr6-15 plants, 50RACE
product corresponding to cleavage at the 30miR390 site, but
not at the secondary site, was detected (Figure 3A; lanes 4–6).
Loss of secondary cleavage in rdr6-15 was expected due to
lack of the complementary RNA strand from which the second-
ary siRNA originates. In zip-1 plants, 50RACE products
corresponding to both miR390-guided and secondary cleavage
events were lost (Figure 3A; lanes 7–9), indicating that AGO7 is
necessary for initiation cleavage of endogenous TAS3a tran-
dual miR171 target-site substitution construct (Figure 2A) was
transiently expressed in N. benthamiana. When expressed alone
or in combination with 35S:MIR171a and 35S:MIR390a, each
construct failed to yield syn-tasiRNA (Figure 3C; lanes 3–12).
But when coexpressed with 35S:MIR171a, 35S:MIR390a, and
35S:HA-AGO7, both the parental construct and the single 30
miR171 target-site substitution construct yielded relatively high
levels of syn-tasiRNA (Figure 3C; lanes 13 and 14). However,
none of the single or dual miR171 substitution constructs with
a 50miR171 target site yielded syn-tasiRNA, even when coex-
pressed with 35S:HA-AGO7 (Figure 3C; lanes 15–17). Cleavage
was detected at each of the 30and cleavable 50miR171 target
sites in the presence and absence of HA-AGO7 (Figure 3D; lanes
3, 5, 6, 8, 10, and 11), indicating that another AGO protein (likely
AGO1, see below) functions in association with miR171. Ectopic
miR171 was not required for cleavageat miR171 target sitesdue
to relatively high levels of endogenous miR171 (Figure 3D; lanes
3, 5, and 6). In contrast to miR171 target sites, processing at the
authentic 30miR390 target site was dependent on coexpression
with 35S:HA-AGO7, irrespective of the corresponding 50target
site (Figure 3D; lanes 7, 9, and 10 versus lanes 12, 14, and 15).
The requirements for AGO7 and miR390 in cleavage and syn-
of AGO7 for cleavage specifically at the 30miR390 target site,
but not at miR171 target sites, suggests that AGO7 functions
as a miR390-specific slicer.
Figure 1. Functionality and Genetic Requirements of TAS3a-Based syn-tasiRNA
(A) Organization of syn-tasiRNA constructs. The miR390-guided cleavage site is indicated by the arrow. The tasiRNA region is indicated by brackets.
(B) Phenotypes of Col-0 and mutant plants containing empty vector, 35S:TAS3aPDS-1, and 35S:TAS3aPDS-2 (Ad = adaxial, Ab = abaxial).
(C) Mean relative level ± SE of PDS mRNA in rosette tissue (Col-0 vector = 1.0).
(D) Mean relative level ± SE of PDS mRNA in leaf midrib and lamina tissue (Col-0 vector = 1.0).
(E) Mean relative level ± SE of syn-tasiRNA (Col-0 35S:TAS3aPDS-2 = 1.0). Inset shows small RNA blot data.
(F) GUS activity in seedlings of Col-0 plants transformed with the indicated constructs.
(G) Organization of HA-AGO7.
1.0) from a transient assay in N. benthamiana.
Table 1. Effects of TAS3a-Based Syn-tasiRNA on Wild-Type and
Vector 90 ?
35S:TAS3aPDS-2 12 ++ 8.316.775.0
35S:TAS3aPDS-1 85 ?
35S:TAS3aPDS-1 12 ?
aSyn-tasiRNA are scored as either present (+) or absent (?) as deter-
mined by RNA blot assays.
bPhotobleaching is shown as a percentage of plants in each category.
cThe PDSd7 (position 50D7[+]) syn-tasiRNA differs between each of the
dThe PDSd8 (position 50D8[+]) syn-tasiRNA is identical in each of the
Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc. 131
AGO7 Associates Specifically with miR390
To determine if AGO7 associates specifically with miR390, an
authentic 50and 30regulatory sequences was introduced into
Col-0 and zip-1 plants and analyzed for small RNA association
in coimmunoprecipitation (coIP) assays. zip-1 plants exhibit
a number of developmental abnormalities, including defects in
leaf development, vegetative timing, and seed set. The
AGO7:HA-AGO7 construct complemented the zip-1 develop-
mental defects and restored TAS3 tasiRNA (Figure 4A). Pre-IP
(input) and anti-HA IP fractions from extracts of zip-1 vector-
and AGO7:HA-AGO7-transformed plants were analyzed for
AGO7, miR171, and miR390. HA-AGO7 was detected only in
the input and IP fractions from zip-1 plants with AGO7:HA-
AGO7. miR171 and miR390 were detected in the input fraction
of both zip-1 vector- and AGO7:HA-AGO7-transformed plants,
but onlymiR390 coimmunoprecipitated
(Figure 4B). Neither miR171 nor miR390 was detected in the IP
fraction from zip-1 vector-transformed plants (Figure 4B; lane 2).
The AGO7-associated smallRNAs were probed more exhaus-
tively by sequencing amplified small RNA populations from
HA-AGO7 input and IP fractions (Figure 4B; flow chart). Small
RNA of the 24 nt size class were most abundant in the input frac-
tion, whereas 21 nt small RNAs were most abundant in the IP
fraction (Figure S3A). Read numbers for previously defined
Arabidopsis miRNA and tasiRNA families were recorded from
Figure 2. Requirement and Specificity of miR390 for Syn-tasiRNA Formation
(A) The 30and/or 50miR390 target sites in 35S:TAS3aPDS-2 were substituted for miR171 target sites. The proportion of cloned 50RACE products corresponding
target site are indicated by gray arrows, although proportions are not indicated. The percentages of plants that displayed photobleaching are shown next to
representative images of rosettes (for each line, n R 5 primary transformants analyzed).
(B) Mean relative level ± SE of syn-tasiRNA (Col-0 35S:TAS3aPDS-2 = 1.0).
132 Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc.
Figure 3. AGO7 and miR390 Requirements for Processing of TAS3a-Derived Transcripts
(A) 50RACE assay of cleavage of endogenous TAS3a transcripts.
(B) Schematic for transient expression assay in N. benthamiana.
(C) RNA blot assays for small RNAs, with mean relative levels ± SEs of syn-tasiRNA shown, and blot images shown for one replicate (35S:MIR171a +
35S:MIR390a + 35S:TAS3aPDS-2 + 35S:HA-AGO7 = 1.0).
(D) 50RACE assay of miRNA-guided cleavage. Mean relative levels ± SEs of 30cleavage product accumulation corresponding to cleavage at the 30miRNA target
sites, with gel images shown for one replicate (35S:MIR171a + 35S:MIR390a + 35S:TAS3aPDS-2 + 35S:HA-AGO7 = 1.0).
(E) Summary of miR390 and AGO7 requirements for cleavage and syn-tasiRNA formation in modified TAS3a-based syn-tasiRNA transcripts.
Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc. 133
Figure 4. Small RNA Interactions with AGO1, AGO2, and AGO7
(A) Wild-type (Col-0) and zip-1 vector- and AGO7:HA-AGO7-transformed plants. Mean ± SE of leaf blade length/petiole length ratios for leaves 1–6 (n R 17 for
each line) are shown in the line graph. Mean relative levels ± SE of TAS3a tasiRNA (Col-0 vector-transformed = 1.0) are shown in the bar graph.
(B) Analysis of Arabidopsis small RNA populations associated with HA-AGO7. Protein and RNA blot assays using input (in) and IP (HA) fractionsare on the right of
the flowchart. Enrichment or depletion of the indicated miRNA and tasiRNA families in the HA-AGO7 IP fraction relative to the input fraction is shown in the lower
134 Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc.
the input (10,226) and IP (14,226) fractions (Table S2). Absolute
read counts as a reflection of AGO7-associated small RNAs in
the IP fraction, however are not directly interpretable due to
the effects of contamination with AGO7-non-associated small
RNA. For example, reads of a highly enriched, but low-abun-
dance, miRNA could be dwarfed by reads from a normally
abundant miRNA that contaminates the IP fraction. Therefore,
small RNA association with AGO7 was assessed by calculating
enrichment in the IP fraction relative to the input fraction. Overall
representation of individual family reads/total miRNA+tasiRNA
reads in IP and input fractions was determined for each family,
and enrichment or depletion in the IP fraction was calculated
using the IP representation/input representation ratio. Only
miRNA or tasiRNA families that were represented in both input
and IP fractions, and by at least five reads in either fraction,
were included in the analysis. miR390 and miR391, which
comprise one miRNA family, were treated independently here
because of their specific relevance to the TAS3 pathway.
The majority of miRNA families were underrepresented in the
IP fraction (Figure 4B). Similarly, each of three tasiRNA families
were underrepresented. In contrast, miR390 was enriched
?28-fold in the IP fraction. miR390 was the only miRNA enriched
more than 3.1-fold (Figure 4B), suggesting that AGO7 has
resented in the IP fraction. Also, several miRNA families were
neither enriched nor depleted in the IP fraction. While this could
conceivably reflect a weak or unstable association with AGO7,
the high endogenous levels for most of these families suggests
it is more likely due to nonspecific contamination.
The apparent preference of AGO7 for miR390 in Arabidopsis
plants could be explained by a unique overlap in expression
domains of AGO7 and MIR390 genes. To eliminate confounding
spatial and temporal effects, competition IP assays were done
using the transient expression system. In three of four assays,
35S:HA-AGO7 or empty vector was coexpressed with 35S:
TAS3aPDS-2, 35S:MIR390a and one or two additional 35S
promoter-driven MIRNA constructs (for miR171, miR159,
miR167, and miR319). In a fourthassay, 35S:HA-AGO7 or empty
vector was coexpressed with 35S:MIR390a, 35S:TAS3aPDS-2,
35S:MIR173a, anda 35S-driven
tasiRNA construct (35S:TAS1cPDS-2). miR390 coimmunopreci-
pitated with HA-AGO7 in each of the competition assays
(Figure 4C, lane 4; only one experiment). In contrast, none of
the other miRNAs or syn-tasiRNAs tested coimmunoprecipi-
tated with HA-AGO7 (Figure 4C; lane 4), strongly supporting
the hypothesis that AGO7 associates specifically with miR390.
miR390 Selectivity for AGO7
The association of AGO7 with miR390 led to the question of
whether or not miR390 is excluded from the other Arabidopsis
AGO proteins that associate with miRNAs. HA-AGO1 and
HA-AGO2 were compared to HA-AGO7 for their association with
miR390, miR171, and miR173 in competition IP assays. AGO1 is
known to associate with most miRNAs, TAS1 tasiRNA, and
some viral siRNA (Baumberger and Baulcombe, 2005; Qi et al.,
2005, 2006; Zhang et al., 2006). Little is known about AGO2 func-
tionality,butitwas chosenbecauseofclose relatednesstoAGO7
within the 10-member AGO family in Arabidopsis (Tolia and
HA-AGO7, or empty vector was coexpressed with 35S:MIR171a
alone or a mixture of 35S:MIR173a and 35S:MIR390a. Consistent
with previous experiments, only miR390 coimmunoprecipitated
with AGO7 (Figure 4D; lanes 5 and 6). Conversely, miR171 and
miR173, but not miR390, coimmunoprecipitated with AGO1
pitated with AGO2, but at a relatively low level, while miR171 and
miR173 did not (Figure 4D; lanes 7 and 8). Using transgenic Col-0
plants expressing HA-AGO2 from authentic regulatory signals
(AGO2:HA-AGO2), small RNAs associated with AGO2 were
sequenced, and enrichment/depletion ratios were calculated.
Small RNAs in the HA-AGO2 IP fraction were primarily 21 nt in
length (Figure S3B). Most miRNAs and tasiRNAs were depleted
in the HA-AGO2 IP fraction, but with a few notable exceptions.
S3). Although the miR390 ratio was neutral, it ranked seventh
among miRNA families. Interestingly, six of the top seven ranking
miRNAs, including miR390, contain 50A residues, which contrasts
with the vast majority of miRNAs, including miR171 and miR173,
which contain 50U. Additionally, the 50A-containing subset of
tasiRNAs from three families were specifically enriched by
HA-AGO2 IP, whereas non-50A-containing tasiRNAs were
depleted. These results hint at a prospective 50rule for specificity
small RNAs containing 50U or 50A, respectively. AGO7 specificity
for miR390, however, may have more complex requirements
than can be explained by a simple 50feature.
Specificity in AGO7-miR390 Complex Formation
and 50Nucleotide Discrimination
To test directly the miR390 50nucleotide requirement, as well as
loading specificity, mutant and chimeric miR390- and miR171-
expressing constructs were analyzed (Figure 5A). As described
above, these experiments were done in the context of AGO1,
AGO2, and AGO7 proteins to understand both the AGO7-
miR390-specific features and the broader principles of AGO-
miRNA complex formation.
We first hypothesized that the strong association of miR390
with AGO7 is dictated by the miR390 50A. We also hypothesized
(C) Protein and RNA blot assays using input (in) and IP (HA) fractions from N. benthamiana following coexpression of empty vector or 35S:HA-AGO7 and 35S:
TAS3aPDS-2 or 35S:TAS1cPDS-2, and various 35S-promoter driven MIRNA constructs.
(D) Protein and RNA blot assays using input (in) and IP (HA) fractions from N. benthamiana following coexpression of empty vector, 35S:HA-AGO1, 35S:
HA-AGO7, or 35S:HA-AGO2 and various 35S-promoter-driven MIRNA constructs.
ranged from ?1.1- to ?255-fold, but the display was limited at ?6.0. Families for which no reads were obtained from one fraction, but for which at least 14 reads
were obtained in the other fraction, are marked with an asterisk.
Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc. 135
that miR171 associates with AGO1 due to a 50U. Parental
35S:MIR171a and 35S:MIR390a were mutated to convert the
50nucleotides of miR171 and miR390 to A and U (forming
35S:MIR171a-50A and 35S:MIR390a-50U), respectively, and
miRNA association with HA-tagged AGO1, AGO2, and AGO7
was assessed in coIP assays (Figure 5A). HA-AGO1 specifically
Figure 5. Specificity Determinants for AGO1, AGO2, and AGO7
(A) Predicted foldbacks of MIR171a and MIR390a mutant and chimeric constructs.
(B) Protein and RNA blot assays using input (in) and IP (HA) fractions from N. benthamiana following coexpression of empty vector, 35S:HA-AGO1, 35S:HA-
AGO7, or 35S:HA-AGO2 and 35S-promoter-driven parental or mutant MIR171a and MIR390a constructs.
(C) RNA blot assays for miR390 and syn-tasiRNA from N. benthamiana assays. Mean relative levels ± SE of syn-tasiRNA are shown with blot images for one
replicate (35S:MIR390a + 35S:TAS3aPDS-2 + 35S:HA-AGO7 = 1.0).
(D and E) Protein and RNA blot assays using input (in) and IP (HA) fractions of leaf tissue extracts from N. benthamiana following coexpression of empty vector,
35S:HA-AGO1, or 35S:HA-AGO7 and 35S-promoter-driven MIR171a, MIR390a, or MIR171a/MIR390a chimeric constructs.
136 Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc.
(miR171 and miR390-50U), but not with 50A-containing miRNAs
(miR390 and miR171-50A) (Figure 5B). In the reciprocal pattern,
HA-AGO2 specifically coimmunoprecipitated with 50A-contain-
containing a 50U (miR171 and miR390-50U) (Figure 5B). These
data reveal the 50nucleotide as a simple specificity determinant
for inclusion/exclusion from AGO1 (50U) and AGO2 (50A) com-
plexes. However, loading of AGO1 with miR390-50U, or AGO2
with miR390, in the absence of AGO7 was insufficient to trigger
syn-tasiRNA formation in coexpression assays with 35S:
TAS3aPDS-2 (Figure 5C; lanes 7 and 10).
In sharp contrast, HA-AGO7 coimmunoprecipitated with
miR390 containing either a 50A or 50U, and failed to associate
with either miR171 or miR171-50A (Figure 5B). Coexpression of
either 35S:MIR390a-50U or 35S:MIR390a with 35S:HA-AGO7
and 35S:TAS3aPDS-2 triggered syn-tasiRNA formation, al-
though syn-tasiRNA levels were lower in the assay containing
35S:MIR390a-50U (Figure 5C; lanes 8 and 9). AGO7, therefore,
neither loads miR390 on the basis of a 50A nor excludes miR171
due to a 50U, and the 50identity has only modest effects on func-
Finally, to determine if the MIR390a foldback contains unique
sequence or structural features for loading miR390 into AGO7,
chimeric MIR171a and MIR390a constructs were tested in
coIP assays with HA-AGO1 and HA-AGO7. In the first series,
two miR171 duplex forms, one with an authentic miR171
duplex-mispair structure (C:C and U:C mispairs, 35S:MIR390a-
171sub2) and the other with a miR390-like duplex mispair
structure (single G:A mispair, 35S:MIR390a-171sub1), were
introduced into the MIR390a precursor in place of the miR390
duplex (Figure 5A). miR171 expressed from these chimeric
constructs accumulated to higher levels than when derived
from the authentic MIR171a context, but coimmunoprecipitated
specifically with HA-AGO1 regardless of the precursor context
(Figure 5D; lanes 7–12). None of the chimeric miR171-generat-
ing precursors yielded miR171 with the capacity to coIP with
HA-AGO7 (Figure 5D; lanes 13–18), indicating that neither the
MIR390a precursor stem-loop backbone, nor the positions of
mispairs within the MIR390a miRNA-miRNA* duplex, are suffi-
cient to direct association with AGO7. In the second series,
two miR390a duplex forms, one with an authentic miR390
duplex-mispair structure (G:A mispair, 35S:MIR171a-390sub2)
and the other with an miR171-like duplex mispair structure
(G:G and C:U mispairs, 35S:MIR171a-390sub1), were intro-
duced into the MIR171a precursor in place of the miR171
duplex (Figure 5A). miR390 was only weakly expressed from
the chimeric MIR171a-based constructs, although miR390
levels from both constructs were above the low endogenous
miR390 levels (2.8- to 4.1-fold; Figure 5E, lanes 9, 13, and
15). miR390 derived from the authentic miR390 precursor, as
well as from both of the chimeric MIR171a-based constructs,
11–16), but not with HA-AGO1 (data not shown). These results
indicate that selective association of AGO7 with miR390 cannot
be explained by a 50nucleotide rule or a foldback-related
structure, but rather point to a specific non-50nucleotide feature
of the miR390 sequence itself.
Using the syn-tasiRNA system, we identified mechanistic
features by which two miR390 target sites function specifically
with AGO7 to route TAS3 transcripts through the RDR6/DCL4
pathway. Cleavage at the 30target site of TAS3a-derived
synthesizing a dsRNA substrate for subsequent processing by
DCL4. Cleavage at the 30miR390 target site requires AGO7,
for heterologous miRNA target sites, indicating that AGO7 is
miRNA guide. The obvious prediction from this result, that AGO7
is uniquely associated with miR390, was confirmed in multiple
coIP assays. Therefore, the functionality of AGO7 at the 30target
site is dictated entirely by its miR390-guided slicer activity.
The TAS3a 50miR390 target-site duplexcontainsrequisite mis-
far less tolerant of target-site substitutions. The dependence on
a 50miR390 target site may reflect several possible requirements.
There may be a requirement for a miR390 guide and/or miR390
target sequence per se, although the unique contribution of the
nucleotide sequences themselves is not obvious. Furthermore,
suggesting that there is a unique requirement for an AGO7-
containing complex at the 50target site. The preference for a 50
of an AGO7-miR390 complex with the precursor transcript. Given
complex is proposed to operate through a ‘‘stable’’ interaction.
How might stable association with an AGO7-miR390 complex
facilitate routing of TAS3a transcripts through the RDR6/DCL4
pathway? One simple model states that AGO7 stabilizes the
processed TAS3 transcript and provides greater opportunity
for interaction with RDR6 (Figure 6A). Alternatively, AGO7 may
actively recruit RDR6 through direct interaction or through asso-
ciated factors, such as SGS3 (Figure 6A). In Arabidopsis, AGO4
interacts with NRPD1b, a subunit of PolIVb (Li et al., 2006;
El-Shami et al., 2007), so there is precedence for AGO proteins
interacting with a polymerase-like protein. A 30end formed by
small RNA-directed cleavage or by other cleavage mechanisms
may be a preferred end for RDR6 activity, possibly due to loss of
the poly(A) tail and associated factors. As another possibility,
stable association of AGO7 might physically direct transcripts
into an RDR6/DCL4-containing processing center (Figure 6A).
Although an RDR6/DCL4 processing center has not been identi-
fied, RNA-processing centers with roles in silencing, such as P
bodies (Liu et al., 2005), have been identified.
Most miRNA target transcripts are not routed through the
RDR6/DCL4 pathway (Axtell et al., 2006; Howell et al., 2007;
dissociation of the AGO-containing complex after cleavage
interaction at single sites. Among the relatively few targets that
feed into the RDR6/DCL4 pathway are several transcripts
encoding pentatricopeptide repeat (PPR) proteins (Axtell et al.,
Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc. 137
2006; Chen et al., 2007; Howell et al., 2007; Lu et al., 2006).
Notably, these are targeted for cleavage by miR161 isoforms
and TAS1/TAS2-derived tasiRNAs, but are also predicted to
be targeted by miR400 (Howell et al., 2007; Peragine et al.,
2004; Sunkar and Zhu, 2004). miR400 fails to direct cleavage
(Howell et al., 2007) and may associate stably with PPR target
transcripts. Thus, the combination of small RNA-directed cleav-
age and noncleaving AGO-containing complexes on a transcript
may provide key signals or structures to efficiently recruit RDR6.
Why is AGO1, which will associate with functional miR390-
50U, unable to substitute for AGO7 at the 50target site? Perhaps
AGO1 fails to recognize or associate stably with more highly
mispaired target sites, whereas AGO7 may be more accommo-
dating of miRNA target mispairs. Alternatively, AGO1 may lack
an AGO7-like ability to efficiently recruit RDR6 to target tran-
scripts. The known role of AGO1 in RDR6-dependent transgene
silencing (Fagard et al., 2000) argues against this idea, although
AGO1 may only be necessary for activity (not biogenesis) of
secondary siRNA. This idea is weakened further by the fact
that TAS1 and TAS2 tasiRNA formation requires RDR6 but not
AGO7. It will be important to learn if other miRNAs involved in
tasiRNA initiation cleavage, such as miR173 and miR828, have
unique AGO associations that facilitate routing through the
How did the specialized interaction between AGO7 and
miR390 in TAS3 tasiRNA formation arise? Given the antiquity
of the TAS3 pathway, it is likely that AGO7 has been finely tuned
through hundreds of millions of years of evolution to recognize
miR390 and exclude other miRNAs. We suggest that AGO7
has subfunctionalized at both the biochemical and expression
levels, the result being only a few miRNA target transcripts
ushered into the RDR6/DCL4 pathway by AGO7. Similarly,
miR390 adopted a 50A feature that excludes association with
AGO1. This 50nucleotide exclusion mechanism should limit
miR390 activity outside of the AGO7 expression domain. Given
the widespread activity of AGO1 as an effector protein for
many posttranscriptional silencing pathways, and the apparent
broad expression domain of MIR390a and MIR390b, the 50
nucleotide exclusion mechanism should focus miR390 activity
to cells that coexpress AGO7. We recognize that, despite the
Figure 6. miR390-AGO Specificity and Three Models for Recruitment of RDR6 to TAS3 Transcripts
(A) 50nucleotide specificity rules apply to AGO1 and AGO2, but not AGO7. Three models (1–3) to explain how the 50miR390-AGO7 complex recruits RDR6 to
TAS3 transcripts are shown in the shaded boxes.
(B) Summary of AGO specificity for miRNA derived from each of the MIR171a and MIR390a 50nt substitution and chimeric constructs (Figure 5A).
138 Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc.
unique affinity of AGO7 for miR390, and the exclusion of miR390
from AGO1, miR390 also associates weakly with AGO2, as well
as with AGO4 (Qi et al., 2006). The functional significance of ei-
ther AGO2 or AGO4 interaction is not understood. Association
of miR390 with AGO2 is clearly driven by the 50A, but the basis
for association with AGO4, which interacts primarily with 24 nt
siRNA (Qi et al., 2006), is not known. The phenotype of AGO4
mutants does not overlap the phenotype of TAS3 tasiRNA-defi-
cient mutants (Zilberman et al., 2003). Additionally, AGO7-,
TAS3-, and RDR6-deficient mutants have similar developmental
defects (Adenot et al., 2006; Hunter et al., 2003; Peragine et al.,
2004), indicating that neither AGO2 nor AGO4 functions redun-
dantly with AGO7.
Although we could not identify a role for the miRNA 50nucleo-
tide as an AGO7 specificity determinant, the 50nucleotide was
clearly defined as an inclusion/exclusion specificity determinant
for both AGO1 and AGO2. AGO1 prefers a 50U and excludes
a 50A, whereas AGO2 possesses the reciprocal preference for
50A and exclusion of 50U. Thus, the selective pressure for a 50U
through AGO1, is now understood. Given the widespread pres-
enceof 50Uin animalmiRNA, aswellas50Uorother 50nucleotide
preferences in other small RNA classes (Ruby et al., 2006),
similar AGO selectivity determinants very likely explain at least
some observed 50nucleotide biases more widely. Structural
studies show that AGO-siRNA 50end interactions are important
for recognition, stability, and fidelity of an active effector
complex (Ma et al., 2005; Rivas et al., 2005). Indeed, the 50termi-
nal base interacts directly through base-stacking interactions
with residues in a highly conserved 50nucleotide-binding pocket
(Ma et al., 2005; Parker et al., 2005). In Archaeglobus fulgidus
Piwi protein, mutation of a tyrosine residue within the 50nucleo-
tide-binding pocket known to interact with the 50base results in
decreased siRNA-binding affinity (Ma et al., 2005). Thus, it is
possible that variation within this pocket accounts for differing
50nucleotide specificities among AGO1, AGO2, and AGO7.
Analysis of the chimeric foldbacks indicated that neither the
MIR390a stem-loop context nor the miR390/miR390* base-pair
and mispair features account for the affinity of AGO7 for
miR390 (Figure 6B). However, small RNA duplex structure
does specify loading for Ago1 and Ago2 in Drosophila. miRNAs
with central mispairs in the miRNA/miRNA* duplex are preferen-
tiallyloaded into Ago1, whereasperfectly paired siRNAduplexes
are preferentially loaded into Ago2 (Forstemann et al., 2007;
Tomari et al., 2007). A similar sorting mechanism exists in
C. elegans (Steiner et al., 2007). Thus, AGO specificity can be
directed by specific 50nucleotide identities as well as base-
pair structure of precursors. The finding that miR390-AGO7
association depends on additional features suggests there are
more specificity determinants to discover.
Finally, the apparent restricted domain of AGO7 promoter
activity in Arabidopsis plants and the ability of ectopic AGO7 to
overcome limitations to syn-tasiRNA formation in N. benthami-
ana leaves suggest that AGO7 may limit activity of the TAS3
photobleaching phenotype appears to emanate from tissue
with AGO7 promoter activity. Expansion of the phenotype
away from vascular cells may reflect cell-non-autonomy of
syn-tasiRNAs. Given that DCL4 products likely function as
mobile silencing signals (Dunoyer et al., 2005), TAS3a-derived
tasiRNAs might function in a cell-non-autonomous manner
during plant growth and development.
Transgene sequences were PCR-amplified from genomic DNA or cDNA
(Supplemental Experimental Procedures). Syn-tasiRNA sequences, miRNA
target-site substitutions, and miR171 and miR390 substitutions were intro-
duced by site-overlap extension PCR (Ho et al., 1989). The resulting products
were cloned in pENTR (Invitrogen), followed by recombination into the plant
transformation vector pMDC32, pMDC163 (a GUS fusion vector), or
pMDC99 (Curtis and Grossniklaus, 2003).
rdr6-15, dcl4-2, and zip-1 alleles were previously described (Allen et al., 2004;
Agrobacterium tumefaciens GV3101 (Clough and Bent, 1998). Transgenic
plants were grown on MS medium containing hygromycin (50 mg/ml) for
7 days, transferred to soil, and maintained in a standard greenhouse with
a 16 hr light/8 hr dark supplemental light cycle.
RNA Blots, Quantitative PCR, and 50RACE Assays
RNA blot assays were done as described (Allen et al., 2005). Except where
noted, triplicate samples from pools of independent primary transformants
were analyzed. Quantitative RT-PCR was done using the same RNA prepara-
tions used for RNA blots. PDS mRNA levels were normalized against ACTIN2
mRNA levels. miRNA-guided cleavage was tested using RNA ligase-mediated
50RACE (Llave et al., 2002) (Supplemental Experimental Procedures).
Transient Expression Assays
Agrobacterium-mediated transient assays in N. benthamiana leaves were
done as described (Llave et al., 2002). Within each experiment, concentrations
of Agrobacterium containing each construct were equalized by adjusting the
concentration of Agrobacterium culture containing empty vector. RNA was
analyzed 48 hr postinfiltration.
ferricyanide, 1 mM potassium ferrocyanide, 16 mM EDTA, 20% methanol, and
1 mg/ml X-glucuronic acid, followed by incubation at 37?C for 3 or 6 hr. Tissue
was cleared in ethanol and photographed with an Olympus SZX12
Immunoprecipitation and subsequent RNA isolation were done using HA
antibody (clone 12CA5, Roche) (Chapman et al., 2004). For immunoprecipita-
tion from Arabidopsis tissue, flower stages 1–12 were used.
Small RNA Sequencing
Flower tissue (stages 1–12) from zip-1 AGO7:HA-AGO7- or Col-0 AGO2:
HA-AGO2-transformed plants was ground in lysis buffer (Chapman et al.,
2004). Cell debris was removed by centrifugation for 10 min at 12,000 g, and
the supernatant was partitioned into input and IP fractions. RNA was isolated
immediately from the input fraction by phenol/chloroform extraction, followed
by ethanol precipitation. HA-AGO7 and HA-AGO2 were immunoprecipitated
using HA antibody and Protein A agarose beads. After removal of an aliquot,
beads weretreatedwith proteinase K. RNA wasisolatedfrom the supernatant,
and amplicons for sequencing were prepared as described (Kasschau
et al., 2007) with the exception of the adaptor sequences and the use of Phu-
done using an Illumina 1G Genome Analyzer.
Cell 133, 128–141, April 4, 2008 ª2008 Elsevier Inc. 139
Statistical analyses were done using S-PLUS (Insightful) and Excel (Microsoft).
For multiple comparisons, Bonferroni adjustments were applied to p value
significance level cutoffs.
Supplemental Data include three figures, three tables, Supplemental Results,
Supplemental Experimental Procedures, and Supplemental References and
can be found with this article online at http://www.cell.com/cgi/content/full/
We thank Bobby Babra, Desiree Boltz, and Amy Shatswell for technical assis-
tance; Chris Sullivan, Scott Givan and Zach Miller for computational assis-
tance; Kristin Kasschau and Mark Dasenko for assistance with sequencing;
and Jim Roberts for stimulating discussions. This work was supported by
grants from NSF (MCB-0618433), NIH (AI43288), USDA-NRI (2006-35301-
17420), NSFC (30325001), and the Monsanto Corporation.
Received: September 5, 2007
Revised: December 21, 2007
Accepted: February 20, 2008
Published online: March 13, 2008
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