Genome-wide identification of transcripts regulated by RNA silencing pathways.
To identify transcripts regulated by the Argonaute proteins, we analyzed expression profiles of Drosophila
Schneider cells (S2 cells) individually depleted of AGO1, AGO2, PIWI, or AUB by using whole-genome oligonucleotide microarrays. To distinguish clearly transcripts whose levels are regulated by the miRNA pathway, we also profiled RNA expression levels in cells depleted of Drosha (10
). We assessed the efficacy of the depletions by Western blotting. Four days after addition of dsRNA, the steady-state expression levels of Drosha, AGO1, AGO2, and AUB had declined to about 10% of the levels detected in untreated cells (Fig. , lanes 4 versus lanes 1). On day 9, the residual levels of the proteins were less than 10% of those observed in control cells (Fig. , lanes 5). Depletion of Drosha, AGO1, or AGO2 also inhibited cell proliferation, confirming the effectiveness of the dsRNAs (see Fig. S1A in the supplemental material). In the absence of specific antibodies against PIWI, we determined the extent of its depletion by RT-PCR (Fig. ). Importantly, AGO2 depletion had no effect on AGO1 or Drosha expression levels (data not shown).
FIG. 1. Expression profiles of Drosophila S2 cells depleted of Drosha or Argonaute proteins. (A) S2 cells were treated with the dsRNAs indicated above the lanes. The effectiveness of the depletions was analyzed by Western blotting with the antibodies indicated (more ...)
For each depleted protein, we obtained two (Drosha, AUB, and PIWI), three (AGO2), or six (AGO1) independent expression profiles from RNA samples isolated on day 9 (see Materials and Methods for a description of the RNA samples). For AGO1, whose depletion leads to more-widespread changes in RNA levels, we also performed a time course and analyzed expression profiles from RNA samples collected on days 3, 5, and 9 of the same knockdown (Fig. ). Total RNA was isolated from mock-treated cells as a reference (control) sample. To identify mRNAs regulated nonspecifically in response to the dsRNA treatment, we examined mRNA profiles in cells treated with green fluorescent protein (GFP) dsRNA (data not shown).
We assigned detectable transcripts to three classes according to their relative expression levels. These were transcripts at least 1.5-fold underrepresented compared to the reference sample, not significantly changed (less than 1.5-fold different from the reference), and at least 1.5-fold overrepresented (Fig. ). We considered a transcript only if it could be assigned to the same class in the two (Drosha, AUB, and PIWI), the three (AGO2), or five of the six (AGO1) independent profiles obtained on day 9 for these proteins. We validated changes in RNA levels for selected mRNAs by Northern blotting (see below; also data not shown).
Fewer than 2% of transcripts showed altered expression in cells depleted of PIWI or AUB (Fig. ; also see Table S1 in the supplemental material). Most of these transcripts have low levels of expression in wild-type cells, and we did not investigate them further. In cells depleted of Drosha, AGO1, or AGO2, between 6% and 18% of transcripts were differentially expressed (Fig. ).
The expression profiles in cells depleted of Drosha or AGO1 (day 9) were significantly correlated (rank correlation coefficient [r] = 0.7) (Fig. ), indicating that depletion of these proteins affects the expression of a common set of RNAs. Some of the RNAs in this set changed levels concordantly in AGO2-depleted cells (Fig. ). In agreement with this, for AGO2 and AGO1 (day 9) profiles, r = 0.7, and for Drosha and AGO2 profiles, r = 0.4, indicating that Drosha, AGO1, and AGO2 regulate the expression levels of common targets.
Depletion of Drosha and depletion of AGO1 lead to similar expression profiles.
To investigate further the similarity of cellular response to the depletion of Drosha, AGO1, or AGO2, we selected mRNAs belonging to specific classes in the Drosha knockdown (at least 1.5-fold over- or underrepresented, respectively) and analyzed their levels in the AGO1 or AGO2 knockdowns. We observed that of the 233 transcripts at least 1.5-fold overrepresented in Drosha-depleted cells, 58% and 16% were at least 1.5-fold up-regulated in the AGO1 (day 9) and AGO2 knockdowns, respectively (Fig. ; also see Table S2 in the supplemental material). Similarly, of the 233 down-regulated RNAs in Drosha-depleted cells, 61% and 16% exhibited the same regulation in AGO1-depleted (day 9) and AGO2-depleted cells, respectively (Fig. ; also see Table S2 in the supplemental material).
FIG. 2. RNAs regulated by Drosha, AGO1, or AGO2. (A) Expression profiles of RNAs at least 1.5-fold over- or underrepresented in the two independent profiles obtained for Drosha (see Table S2 in the supplemental material). Numbers to the right indicate change (more ...)
Likewise, RNAs showing differential expression in AGO1-depleted cells (day 9) had expression profiles similar to those in the Drosha knockdown, although the relative changes in expression levels were more pronounced in AGO1-depleted cells on day 9 (Fig. ; also see Table S3 in the supplemental material). These results indicate that Drosha and AGO1 regulate common targets, in agreement with the role of these proteins in the miRNA pathway (10
). As mentioned above, a subset of transcripts regulated by AGO2 showed similar expression levels in cells depleted of Drosha or AGO1 (Fig. ; also see Table S4 in the supplemental material), suggesting functional overlap between the three proteins.
Predicted miRNA targets are significantly enriched among up-regulated transcripts.
Given the role of Drosha and AGO1 in the miRNA pathway (10
), changes in mRNA levels observed after their depletion are most likely to be caused by the inactivation of this pathway. We therefore investigated whether the transcripts up-regulated in Drosha- or AGO1-depleted cells were among predicted miRNA targets. The overlap between both sets can be used to distinguish between transcripts whose levels are directly or indirectly affected by miRNAs. The Drosophila
genome encodes ca. 100 miRNAs (2
), of which 53 have been cloned (2
) and 39 have unique (nonredundant) seed sequences (i.e., eight most-5′ nucleotides). We tested the enrichment for targets of nonredundant cloned miRNAs predicted by Stark et al. (49
) by using an algorithm based on experimentally derived rules for miRNA target recognition (6
) and found a significant enrichment for predicted miRNA targets among transcripts up-regulated in the Drosha knockdown (P
= 5.8 × 10−24
) and AGO1 knockdown (P
= 3.0 × 10−34
) (see Table S5 in the supplemental material). Interestingly, transcripts up-regulated in the AGO2 knockdown were also significantly enriched in miRNA predicted targets (P
= 1.8 × 10−9
) (see Table S5 in the supplemental material), suggesting that some miRNAs may not discriminate between AGO1- or AGO2-containing RISCs. Targets predicted in other studies were also represented in the list of up-regulated genes (13
). No significant enrichment for predicted targets was found among down-regulated transcripts (P
values of order unity), suggesting that these transcripts represent secondary targets of the miRNA pathway.
Identification of a core set of transcripts regulated by the miRNA pathway.
To identify potential miRNA targets, we generated a list of transcripts up-regulated at least 1.5-fold in the two profiles obtained for Drosha and in at least five of six profiles obtained for AGO1 (day 9). We found 136 mRNAs in this class, representing 2.3% of detectable RNAs (Fig. ; also see Table S6 in the supplemental material). Although the cutoff ratio of 1.5 is low relative to the standard deviation of all detectable spots in the array (see Materials and Methods), the stringent filtering criterion (i.e., regulation in at least seven of eight independent profiles) reduces the likelihood of selecting false positives. Consistent with this, only four of these transcripts changed levels more than 1.5-fold in cells treated with AUB, PIWI, or GFP dsRNA (see Table S6 in the supplemental material). We define these RNAs as core transcripts, whose levels are regulated by the miRNA pathway.
FIG. 3. Core transcripts regulated by the miRNA pathway. (A) Expression profiles of RNAs at least 1.5-fold overrepresented in Drosha-and AGO1-depleted (day 9) cells (core transcripts) (see Table S6 in the supplemental material). Numbers to the right indicate (more ...)
The list of core transcripts includes hid and reaper mRNAs, which are validated miRNA targets (5
). Indeed, we found that both hid and reaper mRNAs were at least twofold up-regulated in cells depleted of Drosha or AGO1 (day 9) (see Table S6 in the supplemental material). Unexpectedly, both hid and reaper were at least 1.7-fold up-regulated in AGO2-depleted cells (see Table S6 in the supplemental material). Furthermore, among the 136 core transcripts, 31 were at least 1.5-fold up-regulated in the three independent profiles obtained for AGO2 (Fig. ; also see Table S6 in the supplemental material). This lends additional support to the hypothesis that some miRNAs may not discriminate between AGO1- or AGO2-containing RISCs.
The miRNAs with the most significant target gene enrichment among the core transcripts were the K-Box miRNAs (i.e., miR-2, miR-13, miR-6, and miR-11 [P of ~10−12 to 10−6]) (Table ). Targets of miR-308, miR-8, and miR-314 were also significantly enriched (P of ~10−9 to 10−6). The enrichment levels for miR-14 (P = 6 × 10−4) and miR-9a and miR-9b (miR-9a/b) (P = 1 × 10−2) targets (Table ) were also significant, although these miRNAs have not been shown to be expressed in S2 cells. Our results suggest that miR-9 and miR-14 might be expressed in S2 cells under our experimental conditions. Indeed, these miRNAs are detectable in S2 cells (Fig. ).
Enrichment of predicted miRNA targets among core transcripts
Analysis of the biological function of the proteins encoded by core transcripts done using gene ontology terms (3
) revealed that some functional groups are overrepresented in the list of core transcripts in comparison to the detectable transcripts (Fig. ; also see Table S6 in the supplemental material). In particular, we observed a significant enrichment of genes involved in developmental processes (P
= 5 × 10−3
), axonogenesis (P
= 4 × 10−3
), organogenesis (P
= 7 × 10−3
), cell adhesion (P
= 1 × 10−2
), and signal transduction (P
= 1 × 10−2
Compared to the distribution of abundance of detectable transcripts, core transcripts show a bias towards low abundance in wild-type cells but an almost normal distribution in AGO1-depleted cells (Fig. ), suggesting that these transcripts are not intrinsically of low abundance but rather are down-regulated by the miRNA pathway in wild-type cells.
Core transcripts represent authentic miRNA targets.
To investigate whether predicted miRNA targets in the list of core transcripts represent authentic targets, 3′ UTRs derived from eight core transcripts were cloned into a firefly luciferase sensor reporter (42
). We selected transcripts that were also regulated by AGO2. Four of them were predicted miR-9a/b targets. A previously validated miR-9b target, Nerfin (49
), served as the positive control. When cotransfected with at least one of the predicted cognate miRNAs, six of eight of the 3′ UTRs led to a reduction of luciferase activity (relative to the activity observed in the absence of the miRNA) (Fig. ).
FIG. 4. Core transcripts represent authentic miRNA targets. (A and B) Reporter plasmids constitutively expressing firefly luciferase (luc.) flanked by the 3′ UTRs of predicted miRNA targets and plasmids expressing miRNA primary transcripts were cotransfected (more ...)
We found that predicted miR-9 targets were often regulated exclusively by either miR-9a or miR-9b (Fig. ) (e.g., CG10011 and Nerfin), indicating that these miRNAs are not redundant, despite their sequence similarity. Also, for some reporters (e.g., CG4851, Sema-1b, and CG12505) coexpression of an miRNA led to an increase in luciferase protein expression (Fig. ). One possible explanation for these results is that these miRNAs silence the expression of a negative regulator.
The results described above raised the question of whether predicted miRNA targets not included in the list of core transcripts also represent authentic targets. We therefore tested two 3′ UTRs derived from transcripts (CG30337 and CG33087) that were regulated in Drosha- and AGO1-depleted cells but were not in the list of core transcripts because they were not detectable in two experiments. These reporters were also down-regulated by at least one of the miRNAs predicted to recognize these 3′ UTRs (Fig. ). This observation confirms the assumption that the filtering criterion to select core transcripts (regulation in seven of eight independent profiles and detectable in all profiles) is stringent and that some genuine targets are excluded.
We also selected nine 3′ UTRs from predicted targets of miR-9a/b, miR-13a/b, and miR-14 whose expression levels remained unchanged in depleted cells and were comparable to those of the core transcripts in wild-type cells. Four out of nine 3′ UTRs tested repressed luciferase expression in the presence of the cognate miRNA (Fig. ). Note that for these 3′ UTRs we have not tested all miRNAs predicted to have binding sites, so the fraction of these transcripts representing authentic miRNA targets is likely to be underestimated.
We conclude that although the majority of predicted miRNA targets in the list of core transcripts are genuine targets of the miRNA pathway, this list is not comprehensive and additional targets may be identified when less stringent criteria are applied. Furthermore, not all miRNA targets are subject to down-regulation of mRNA levels, and some miRNA targets might not be regulated at all in S2 cells.
AGO2 associates with miRNAs.
In a previous study, we showed that expression of firefly luciferase from the reporters harboring Vha68-1 or CG10011 3′ UTRs in the presence of miR-9b or miR-12 could be restored in cells depleted of AGO1 but not of AGO2 (42
), despite Vha68-1 and CG10011 mRNA levels being regulated in AGO2-depleted cells. We obtained similar results for the reporter containing the Nerfin 3′ UTR (Fig. ). Depletion of Drosha also led to a partial restoration of firefly luciferase expression from these reporters, providing further evidence for a regulation of these reporters via the miRNA pathway (Fig. ). The lack of restoration in AGO2-depleted cells is not caused by an inefficient depletion, because silencing of firefly luciferase expression by cotransfecting a fully complementary siRNA (Luc-siRNA) is impaired in these cells (see Fig. S1B in the supplemental material) (42
). Thus, depletion of AGO2 inhibits siRNA-guided but not miRNA-guided gene silencing, as reported by Okamura et al. (36
These results contrast with the observation that AGO1 and AGO2 regulate the expression levels of a common set of miRNA targets. We therefore reasoned that regulation by AGO2 may not be observed with the reporter assays described above, as in this case both the reporter and the miRNAs are overexpressed. To investigate whether AGO2 associates with endogenous miRNAs, we performed immunoprecipitations from total lysates of S2 cells expressing a HA-tagged version of AGO1, AGO2, or MBP as a control. The presence of miRNAs associated with the precipitated proteins was analyzed by Northern blotting. Although the expression levels of these proteins were comparable, HA-tagged AGO1 immunoprecipitated very inefficiently (Fig. ). Nonetheless, miR-13b and bantam coimmunoprecipitated with HA-tagged AGO1 (Fig. ). HA-tagged AGO2 also immunoprecipitated these miRNAs above background levels, indicating that a small fraction of endogenous miRNAs can be found in association with AGO2 (Fig. ). These results provide an explanation for the observation that a subset of miRNA targets is regulated by AGO2.
FIG. 5. AGO2 associates with miRNAs. (A) Immunoprecipitation of HA-tagged AGO1, AGO2, or MBP from total cell lysates. The right panel shows a longer exposure of the immunoprecipitated samples to visualize the presence of AGO1. α-HA, anti-HA. (B) The presence (more ...) A few transcripts are regulated exclusively in the individual knockdowns.
To determine whether Drosha, AGO1, and AGO2 have evolved specialized functions, we searched for transcripts regulated exclusively in one of the knockdowns but clearly unaffected (less than 1.3-fold) or showing inverse correlation in the other knockdowns. Only four transcripts were found to be regulated exclusively in Drosha-depleted cells (Fig. ). It would be of interest to determine whether Drosha regulates the expression of these transcripts by a mechanism not involving miRNAs.
FIG. 6. RNAs regulated exclusively in the individual knockdowns. RNAs regulated exclusively by Drosha (A and B), AGO1 (C), or AGO2 (D and E) showing noncorrelated expression in the other knockdowns. (F) RNAs regulated exclusively in the four profiles obtained (more ...)
We were also able to identify transcripts regulated by AGO1 but not by Drosha or AGO2 and transcripts regulated by AGO2 but not by Drosha or AGO1 (Fig. ). The latter would be explicable if AGO2 regulates the expression of these transcripts by a mechanism involving, for example, siRNAs that are not processed by Drosha.
We also detected transcripts regulated by Drosha and AGO1 but unaffected in AGO2-depleted cells (Fig. ) or transcripts regulated by AGO1 (showing correlated expression in AGO2-depleted cells) but unaffected by Drosha depletion (not shown). Finally, we noticed that Dicer-1 and Dicer-2 mRNAs were at least 1.5-fold up-regulated in AGO1-depleted cells (in four of six profiles). Drosophila
Dicer-1 mRNA has one target site, for miR-314, and Dicer-2 mRNA has sites for miR-280 and miR-315 (note that these sites are not conserved in D. pseudoobscura
). This suggests that a feedback mechanism regulates the expression of genes involved in RNA silencing. Similarly, expression of Dicer-like 1 (DCL1) is regulated by miR-162 in Arabidopsis thaliana
) and expression of AGO1 is regulated by miR-168 (50
Among the transcripts regulated exclusively by AGO2 depletion, we found the transposable element (TE) blood (Fig. ). This prompted us to investigate whether additional transposon-derived transcripts are regulated in depleted cells. There are 96 families of transposable element in Drosophila
, which represent 22% of the genome (19
). TEs are represented by 85 probe sets on the array, most of which correspond to long terminal repeat and non-long terminal repeat retrotransposon families. We found that 21% and 41% of detectable TEs were at least 1.5-fold up-regulated in cells depleted of AGO1 or AGO2, respectively (see Fig. S1C and D and Tables S3 and S4 in the supplemental material). With two exceptions, transposons up-regulated in cells depleted of AGO1 were also up-regulated in the AGO2 knockdown, providing further evidence for functional cross talk between these proteins.