miRNA* species with modified 3′ ends
We previously reported that many miRNA* strands are present in AGO1 complex in Drosophila
embryos and cultured cells (Okamura et al., 2008b
). However miR-276a* and miR-184* caught our attention because they co-precipitated poorly with AGO1, relative to their amount in total RNA. Such behavior might be explained by atypically slow unwinding of their precursor duplexes during maturation of the AGO1 complex. Alternatively, these miRNA* strands might be located in a complex other than AGO1. To distinguish these possibilities, we checked for 3′ modification. HEN1 methyltransferase modifies single stranded siRNAs in AGO2 and piRNAs in PIWI-class Argonaute complexes, which therefore carry 2′-O-methylation at their 3′ ends (Horwich et al., 2007
; Saito et al., 2007
). In contrast, double-stranded duplexes and mature miRNAs in AGO1 complex have predominantly free 2′ and 3′ –OH at their 3′ ends.
The β-elimination assay increases mobility of small RNAs with free 2′ and 3′ –OH group at their 3′ termini (Hutvagner et al., 2001
). We observed that the miRNAs bantam, miR-276a and miR-184 all exhibited increased mobility following this treatment (), supporting the previous conclusion that a majority of the pool of mature Drosophila
miRNAs have free 3′ ends and sort to AGO1. We also tested the endo-siRNAs hp-CG4068B and G, which are known to complex with AGO2, and they were resistant to β-elimination as previously reported (Kawamura et al., 2008
; Okamura et al., 2008a
). When we analyzed miR-276a* and miR-184*, which were not efficiently co-precipitated with AGO1 (Okamura et al., 2008b
), we found that they were nearly completely resistant to β-elimination like endo-siRNAs (). Bantam* was similarly β-resistant. Therefore, some small RNAs derived from Drosophila
miRNA genes are efficiently modified at their 3′ termini.
miRNA* species that are preferentially sorted into AGO2 complex
Because small RNAs in AGO2 complex are modified at their 3′ ends (Horwich et al., 2007
; Pelisson et al., 2007
), we tested whether these miRNA* species associated with AGO2. To do so, we generated S2-R+ cells that were stably transfected with FLAG-HA-AGO2 plasmid (Czech et al., 2008
). These cells slightly overexpressed AGO2 protein, as judged by Western blotting using anti-AGO2 and anti-FLAG antibodies (), but the moderate increase in AGO2 did not substantially affect the steady levels of endo-siRNAs or miRNAs in these cells (, see input lanes). Therefore, these cells should represent an appropriate setting to study small RNA sorting.
We purified AGO2 and AGO1 complexes from these cells using anti-FLAG and anti-AGO1 antibodies. Consistent with previous reports, endo-siRNAs and miRNAs were highly enriched in AGO2 and AGO1 complexes, respectively (). In contrast to their partner mature miRNAs, miR-276a*, miR-184* and bantam* were highly enriched in AGO2 complex, with enrichment comparable to that of endo-siRNAs (). Together with the β-elimination experiments, we conclude that the star strands of mir-276a, mir-184, and bantam are preferentially sorted into AGO2 like endo-siRNAs, even though they derive from the miRNA pathway. Notably, the specific incorporation of only one duplex strand into AGO2 was not observed in previous studies. For example, the two strands of the endo-siRNA duplex hp-CG4068B/G accumulate asymmetrically, but both strands are sorted to AGO2 (). Therefore, the rules for strand selection by different Drosophila Argonautes are more complex than previously anticipated.
The accumulation of AGO2-loaded miRNA* species requires RNAi factors
In light of the efficient loading of many miRNA* species into AGO2, we tested whether their biogenesis required miRNA or RNAi factors. We depleted a panel of small RNA biogenesis factors by soaking aliquots of S2R+ cells with cognate dsRNAs for 8 days. We observed reduction of mature miRNAs (bantam) and hpRNA-derived siRNAs (hp-CG4068B) upon knockdown of the expected miRNA or RNAi factors (Czech et al., 2008
; Kawamura et al., 2008
; Okamura et al., 2008a
), indicating their functional depletion ().
Distinct biogenesis and activity of miRNA and miRNA* species
In contrast to mature miR-276a and miR-184, their partner miRNA* species were sensitive not only to loss of the miRNA factor Dcr-1, but also to loss of the canonical RNAi factor Dcr-2 (, “Dcr-2”-boxed lanes). In addition, we observed that these miRNA* species exhibited specific dependence on AGO2 instead of AGO1 for normal accumulation, consistent with the biochemical evidence for the residence of these miRNA* species in AGO2 (). However, the accumulation of miRNA* species was more similar to miRNA strands in that they lacked the strong requirement for loqs observed for most endo-siRNAs (, hp-CG4068B blot). In summary, the biogenesis of these miRNA* species depends on a unique combination of canonical miRNA and RNAi factors, not previously observed for mature miRNAs or endo-siRNAs.
AGO2-loaded miRNA* species are poorly active on seed targets
Unlike AGO1 complex that can suppress target expression by seed base-pairing, AGO2 was suggested to be poorly active on target mRNAs with imperfect complementarity to guide small RNAs (Forstemann et al., 2007
). Because miR-276a* and miR-184* were preferentially sorted to AGO2 complex, one might predict that these small RNAs would fail to suppress bulged targets as efficiently as canonical miRNAs.
We tested this using luciferase sensors with perfectly complementary target sites or target sites with central bulges (). To avoid possible mis-sorting of overexpressed miRNA hairpins, we measured the activity of endogenous miRNA and miRNA* species by introducing 2′-O-methyl antisense oligo nucleotides (2Ome-ASOs) and assaying for sensor de-repression. As shown in a previous report (Okamura et al., 2008b
), antisense inhibitors of mature and star strands of miR-276a were able to de-repress both cognate perfect targets confirming that both strands of the duplex are active in S2 cells (). The bulged sensor for the mature strand of miR-276a was also de-repressed by cognate ASO, but further mutation of its seed match resulted in a complete loss of target regulation, as expected for a typical miRNA. We constructed a similar set of sensors for miR-184, which was similarly active on perfect and bulged targets, but not on a bulged target with seed mismatches (Supplementary Figure 1
In contrast, 2Ome-ASOs against miR-276a* did not substantially de-repress bulged sensor activity (). miR-184* similarly showed little activity on its bulged sensor, even though clear de-repression of its perfect sensor by 2Ome-ASO was observed (Supplementary Figure 1
). These results are not simply due to different levels of mature and star strands, at least in the case of mir-276
, since perfect sensors of both mir-276a
strands were similarly de-repressed by cognate inhibitors. We conclude that there is a functional consequence of miRNA* sorting to AGO2, namely the restriction of their target activity on seed-type binding sites. We note that translational repression by AGO2-RISC was recently recognized using in vitro extracts (Iwasaki et al., 2009
). Lack of substantial translational regulation by miRNA*-programmed AGO2 in our cell-based assay might be explained if translational repression by AGO2 is reversible and relatively weaker (Iwasaki et al., 2009
), while AGO1 mediated repression is usually associated with deadenylation and mRNA destabilization.
The strands of synthetic miRNA/miRNA* duplexes can be independently sorted
The cloning of small RNAs from total RNA inevitably yields a variety of related sequences, whose relationship to the RNAs detected by hybridization cannot be explicitly assigned. Conceivably, different cleavage positions of miRNA precursors by Drosha or Dcr-1 might yield a mixture of duplexes with distinct strand selection and/or sorting preferences, which would confound the interpretation of independent sorting of strands from any specific miRNA/miRNA* duplex.
To exclude this possibility, we developed an in vitro assay using synthetic small RNA duplexes (). We labeled the 5′ end of mature miR-276a or star with 32P, and then annealed them to unlabeled partner strands. The labeled duplexes were incubated with lysate prepared from S2-R+ cells expressing FLAG-HA-AGO2 for one hour. AGO2 and AGO1 complexes were then sequentially purified using anti-FLAG and anti-AGO1 antibodies, and the immunoprecipitated small RNAs were analyzed on native acrylamide gels. Although the input RNAs were completely double-stranded, the co-purified small RNAs were predominantly single stranded. Since the labeled RNA recovered from supernatant were mostly double stranded (, “supernatant” lanes), we conclude that single stranded small RNAs co-precipitated with Argonaute complexes represent genuine small RNAs in mature complexes, as opposed to non-specific interactions of fortuitously unwound RNAs to sticky Argonaute proteins.
Distinct loading preferences of the strands of synthetic miRNA duplexes
Under this experimental protocol, unwound miR-276a* strand co-precipitated more efficiently with AGO2 than its partner mature strand (). On the other hand, AGO1 preferred the miR-276a strand (), consistent with in vivo results (). We confirmed this result using a synthetic miR-184/miR-184* duplex, for which AGO1 and AGO2 also exhibited different strand preference (Supplementary Figure 2
). These experiments demonstrate that the strands of defined small RNA duplexes can be independently sorted to different Argonaute complexes.
The influence of 5′ nucleotide on strand selection by AGO1 and AGO2
In plants, the 5′ nucleotide of a small RNA is not only strongly correlated with the choice of Argonaute host, but can actively determine its sorting (Mi et al., 2008
). Animal mature miRNAs exhibit a substantial preference for 5′ uridine, whereas miRNA* species do not exhibit this bias. Largescale sequencing of Drosophila
AGO1- and AGO2-associated RNAs verified that the former are highly enriched in 5′ U, while the latter are not (Czech et al., 2008
), with certain populations of AGO2-resident RNAs exhibiting 5′ C bias (Ghildiyal et al., 2008
). Therefore, one might hypothesize that AGO1 simply prefers RNAs with 5′ U and that AGO2 selects against such RNAs. This might be plausible since the mir-276a
loci we studied in depth, like most miRNAs, encode 5′ U only on their mature strands.
We performed in vitro loading assays of a mir-276a duplex in which the mature strand was altered to 5′ A, while maintaining its structure. The strands of this variant duplex were still reciprocally sorted (), indicating that the lack of 5′ U was not sufficient to promote sorting of miR-276a to AGO2, nor did AGO2 lose its ability to distinguish the star strand when both duplex RNAs bore 5′ A. Similarly, a variant duplex in which both duplex strands bore 5′ U maintained its reciprocal sorting (). However, when we simultaneously changed mature miR-276a to 5′ A and miR-276a* to 5′ U, we reproducibly observed a reversal in the strand selection by AGO1, which now showed preference for the star strand (). Still, AGO2 preferred the star strand of this variant duplex. In summary, strand selection by AGO1 can be dominantly altered by 5′ nt identity, without changing relative end energies, and the strand preference of AGO2 is not adequately explained by either duplex end energy or by lack of 5′ U.
Distinct preferences of AGO1 and AGO2 with respect to miRNA and star strands
Based on our detailed analyses of selected miRNAs, we wished to determine how broadly the principle of independent sorting of miR/miR* strands applies in Drosophila
. Hannon and colleagues reported largescale data on small RNA sequences obtained from AGO1 and AGO2 complexes immunoprecipitated from S2-NP cells (Czech et al., 2008
). In order to obtain an appropriate baseline for interpreting the relative enrichment of depletion of small RNA reads in these IP libraries, we analyzed the total small RNA content of S2-NP cells using Illumina sequencing. We prepared 18–28 nt RNAs and generated 4,310,254 small RNA reads that perfectly matched the Drosophila melanogaster
genome release 5. We then isolated reads that matched the miRNA and miRNA* strands of miRbase version 11 from the S2-NP total RNA, S2-NP-AGO1 and S2-NP-AGO2 libraries (Supplementary Table 1
Our S2-NP total RNA dataset contained 3,819,484 reads matching 100 known miRNA loci, with the content of individual genes ranging over 6 orders of magnitude. We focused our analysis on genes with >100 miRNA reads and >10 miRNA* reads in this dataset. The star read requirement was instated so that inferred miRNA:miRNA* ratios were based on a reasonable sampling of data; however, few (8/46) genes failed this criterion due to our deep library coverage. We then compared the miRNA:miRNA* ratios in total RNA with those obtained from AGO1- and AGO2-IP libraries.
If AGO1 and AGO2 had identical strand preference, we would expect most values to be ~1, with deviations from 1 being due to either cloning/sequencing bias or perhaps small RNAs not in mature complexes. However, we instead saw that most miRNAs were enriched in AGO1 while a majority of miRNA* species were enriched in AGO2. This indicated distinct strand preferences of miRNA duplexes by the two Argonautes. When plotted on a gene-by-gene basis, ~2/3 of miRNA genes exhibited enrichment in AGO2 relative to total RNA (). Collectively, we observed significantly different distribution patterns between AGO1 and AGO2 (), with AGO2 in S2-NP cells carrying more miRNA* reads than miRNA reads ( and Table S2
). Note that some genes exhibiting star-enrichment in AGO2 had more partner miRNA reads in AGO2, because the number of miRNA reads was often much greater than miRNA* reads (Supplementary Table 1
). For example, even though there were 7335 reads of mature bantam and 2238 reads of bantam* in AGO2, this represented ~50 fold enrichment of bantam* in AGO2 in light of the fact that bantam* represents <0.63% of mature bantam in total RNA. We observed similar enrichment of miRNA* strands in AGO2-IP data from female ovaries () (Czech et al., 2008
), indicating that this is a genuine feature of miRNA* sorting in the animal.
Distinct miRNA duplex strand preferences of AGO1 and AGO2
The IP assay by itself does not distinguish between immature duplexes in Argonautes and functionally matured RISC. This may be tested using beta-eliminated libraries, since Hen1 modifies single stranded species in AGO2 (Horwich et al., 2007
). The beta-elimination reaction impairs standard 3′ linker ligation, thereby depleting for AGO1-loaded species and enriching for methylated miRNA or miRNA* species in AGO2. Indeed, we observed a strong enrichment for miRNA* species in S2 beta-eliminated RNA data reported by Zamore and colleagues (Seitz et al., 2008
) ( and Table S2
). In summary, these largescale analyses demonstrate that AGO1 and AGO2 exhibit distinct preferences for the partner strands of most miRNA duplexes, and that a general attribute of AGO2 is the preferred selection of miRNA* strands relative to AGO1.
The pairing status of nucleotides 9 and 10 correlates with sorting profiles
Although strand selection by miRISC and siRISC were thought to be governed by the same thermodynamic rule (Khvorova et al., 2003
; Schwarz et al., 2003
; Tomari et al., 2004
), our genomewide analyses clearly demonstrated distinct loading preferences of partner miRNA and miRNA* species into AGO1 and AGO2. To search for additional factors that influence strand selection and sorting, we analyzed the pairing status across all miRNA/miRNA* duplexes; G:U pairs were considered as a distinct category. To ensure that their companion sorting ratios were meaningful, we used only those miRNA or star sequences with >30 reads in total S2-NP data and >10 AGO1- and AGO2-IP reads; in total, 46 mature strands and 32 star sequences.
As expected, the most substantial difference between the collective pairing status of miRNAs and miRNA* species occurred at the 5′ nucleotide, reflecting the strong influence of thermodynamic asymmetry on strand selection by AGO1 ( and Table S3
). The second-most substantial difference occurred at position 9 from the 5′ end, with unpaired or G:U status correlating with AGO1 loading and paired status correlating with AGO2 loading. We analyzed pairing status with respect to the degree of enrichment in AGO1 or AGO2. This clearly showed that, by far, the pairing status of the 9th position was most correlated with differential sorting into the Argonautes; the 10th position also showed a limited correlation that was evident in this largescale analysis ( and Table S3
Mispairing at positions 9 and 10 correlates with sorting profiles
Pairing at nucleotides 9 and 10 actively determines strand selection by AGO2
The observation of 9th position as a sensitive point for differential AGO loading of miRNA strands was reminiscent of previous observations that central mismatches of positions 7–11 strongly determined AGO partitioning of model duplexes (Tomari et al., 2007
); in fact, greatest sensitivity was observed at position 9. However, the effect of central structure on duplex strand preference was not determined in that study. We directly tested this using the in vitro sorting assay. We first analyzed the sorting of a miR-276 duplex in which the order of bulged positions was reversed with respect to the endogenous duplex (). Interestingly, AGO1 still preferred the mature strand of this variant duplex, but the preference of AGO2 was completely reversed and now loaded only the mature strand. Therefore, the strand selection of AGO2 is very sensitive to internal duplex structure, and its loading properties can be altered without affecting those of AGO1.
AGO2 strand selection is directed by duplex structure at positions 9/10
We probed this further with additional variants. In the endogenous miR-276a duplex, the mature strand is unpaired at positions 9, 10 and 16, whereas the star strand is unpaired at positions 5, 11 and 12. To test whether a seed bulge was relevant for AGO2 strand selection, we reversed the miR-276a* seed bulge to be present in the mature strand; however, this maintained preferred star sorting to AGO2 (). To test whether this was attributable to the position of the centrally paired nucleotides, we reversed the position of the central bulges (). This duplex showed complete selection of the mature strand by AGO2. Given that position 9 showed the greatest correlation for differential miRNA duplex sorting, we tested a miR-276a duplex variant with a single star mismatch at position 9, maintaining a paired 10th position (). This also yielded predominant selection of the mature strand by AGO2. Finally, as our computational analysis suggested that 9th position G:U pairing might be functionally similar to mispairing with respect to AGO2 strand selection, we tested an additional variant. Indeed, introduction of a single G:U pair at position 9 of miR-276a* resulted in nearly complete selection of miR-276a by AGO2 (). We conclude that Watson-Crick pairing at position 9 can actively dictate strand selection by AGO2, and that G:U or mispairing at this position are inhibitory to AGO2 strand selection.
We recognize that the end energies of mir-276a
duplex are relatively similar, so that this particular duplex might in principle be especially sensitive to central structure. We therefore wished to confirm our results with a different miRNA duplex. Zamore and colleagues reported mir-277
as an unusual locus for which a substantial amount of mature strand is sorted to AGO2 (Forstemann et al., 2007
); this duplex is clearly asymmetric with the 5′ end of mature miR-277 residing in the less-structured end. We subjected mir-277
duplex to in vitro sorting assay, and confirmed that AGO2 selected mature miR-277 exclusively. Inspection of this duplex showed that the miR-277* strand has G:U at position 9 (), consistent with rejection of the star strand by AGO2. We tested this by mutating the miR-277* strand to reverse its pattern of central bulges, now paired at 9th and 10th positions of the star strand and unpaired at 9th and 10th positions of the mature strand (). This reversed the behavior of AGO2, which now accepted a substantial amount of miR-277*. This reciprocal redirection strongly supports our proposal that AGO2 strand selection is actively directed by the duplex pairing status of positions 9 and 10, even in the context of a thermodynamically asymmetric duplex.
Studies of siRNA biogenesis indicated that the star strand of the precursor duplex represents the first target of guide strand-programmed cleavage. This is functionally relevant since the introduction of non-cleavable phosphorothiorate (PS) linkages at the scissile phosphodiester bond (Schwarz et al., 2004
) impedes RISC maturation (Leuschner et al., 2006
; Matranga et al., 2005
; Miyoshi et al., 2005
; Rand et al., 2005
). We tested whether passenger strand cleavage was similarly important for maturation of miRNA*-loaded AGO2 complex by introducing PS linkage between nucleosides 10 and 11 of mature miR-276a, thereby blocking its potential cleavage by miR-276a*. However, unwound miR-276a* still accumulated efficiently in AGO2 (Supplementary Figure 3
). This suggests that passenger strand cleavage of miRNA duplexes is not absolutely required for the maturation of miRNA*-loaded AGO2-RISC.
Central duplex structure is less critical for strand selection by AGO1
It is relevant to note that in none of these manipulations, which redirected AGO2 preference from star to mature (for mir-276) or from mature to star (for mir-277), did we observe a reversal of strand selection by AGO1. Moreover, it was clearly evident that while pairing at positions 9/10 are critical for star strand selection by AGO2, pairing at these positions of the mature miRNA was not reciprocally unfavored for loading into AGO1. For example, in the mir-276a duplexes in all are fully paired at the 9th and 10th positions of mature miR-276a. Nevertheless, mature miR-276a was selected by AGO1 from all of these duplex variants, as in the endogenous situation where miR-276a contains G:U 9 and mismatch 10. In addition, full pairing at positions 9 and 10 of mature miR-277 (), as well as full mismatching at these positions (), were both compatible with nearly complete selection of mature miR-277 by AGO1. We infer from these data that AGO1 selects its strand primarily on the basis of the thermodynamic rules and on 5′ nucleotide, and is less actively influenced by central duplex structure.