Photo-crosslinking followed by Ago2 immunoprecipitation, Ago2-CLIP, was used to identify miRNA binding sites in mESCs. We found significantly enriched motifs in 3′UTRs and CDS that correspond to miRNA seed matches, representing 201 and 103 potential mESC miRNA targets in 3′UTRs and CDS, respectively. In regards to the latter point, this study is in agreement with other studies that the presence of miRNA binding sites in CDS is more widespread than has been previously considered and nearly as prevalent as in 3′UTRs14-16
. Here we provided gene expression data suggesting that these CDS sites regulate mRNA stability much like 3′UTR sites. Moreover, these sites can be recognized by miRNAs at endogenous expression levels and confer repression in a heterologous 3′UTR8,13,41,42
Two other Ago-CLIP studies have identified potential miRNA targets in mammalian cells and tissue. Our study differs from those by analyzing mRNAs associated with endogenous Ago2 in a mostly homogenous cell population of mESCs, whereas Chi et al.15
performed CLIP on brain extracts using endogenous Ago antibodies and Hafner et al.14
performed CLIP in 293 cells using HA-tagged Ago1-4 and crosslinking by a photoactivatable nucleotide. Independently of the variations in the CLIP technique and data analysis, these studies, as well as our studies, identified similar numbers of targets for each miRNA seed family (several hundred), which is comparable to the number of moderately conserved targets predicted for each miRNA seed family by TargetScan11
for cross-comparison with other CLIP datasets).
There are several previously published reports of miRNA-regulated mRNAs in mESCs that we could compare to the Ago2-CLIP 3′UTR GCACUU-containing genes35, 43
. Only miR-294 (member of AAGUGC seed family) regulated mRNAs described by Melton et al.43
showed significant overlap with the Ago2-CLIP 3′UTR GCACUU-containing mRNAs (Supplementary Table 6b
for all comparisons).
Unlike other cell types, including those used in other Ago2-CLIP studies14,44
, mESCs appear to be dominated by a single miRNA seed family that is probably responsible for most of the miRNA regulation in this cell type. Essentially all of the GCACUU-motif containing CLIP 3′UTR clusters conferred miRNA-dependent regulation when tested in luciferase reporter assays in the presence of endogenous levels of miRNAs. This suggests that the stringency of our filtering criteria resulted in selection of a high confidence set of GCACUU-containing mRNAs that most likely are bona fide
miRNA targets in mESCs. Previous studies have already shown that this miRNA family plays important roles in mESCs, including maintaining pluripotency, self-renewal and cell cycle control33-35,43,45
. But, few targets have been identified and validated. This study identifying a few hundred new miRNA targets by Ago2-CLIP is a significant step in the exploration of this biology.
To understand the extent of miRNA-regulated pathways represented by the Ago2-CLIP 3′UTR GCACUU-motif genes (“All” set, 201 genes), we performed pathway enrichment analysis (Supplementary Methods
) and compared this set with the top 201 “Conserved predicted targets” and all mRNAs expressed in mESCs that contain GCACUU hexamer in the 3′UTR (“All predicted targets”, 2969 genes). 37 and 11 pathways were significantly enriched in the CLIP and “Conserved predicted targets” sets, respectively (Supplementary Figure 6a
and Supplementary Table 7
). The pathways significantly enriched in CLIP included “Early S-phase” (4 genes), a pathway in which miR-290~295
has been previously implicated33
, and “TGF-beta receptor signaling” (5 genes), a pathway where miR-290~295
has not been implicated.
The genes identified by Ago2-CLIP in “TGF-beta receptor signaling” pathway (p-value 0.013) include two intracellular pathway inhibitors, the cytoplasm-localized Smad7
and the nucleus-localized Skil
, and an extracellular inhibitor, Lefty146
. Our reporter assay confirmed that these 3 genes are indeed targeted by miR-295 (Supplementary Fig. 6b
). We extended this analysis to Lefty2
, a gene that was not identified in the CLIP results, but is homologous to Lefty1
and contains the GCACUU hexamer, and showed that it is also targeted by miR-295 (Supplementary Fig. 6b
). Correlated with this, miR-302
, which are related in miRNA seed to miR-290~295
, have been shown, respectively, in human ESCs47
and zebrafish embryos48
to regulate differentiation through targeting Lefty
homologs. Here using a genome-wide approach, we found that the miR-290~295
regulates not only the extracellular Lefty
homologs, but also additional inhibitory nodes of the TGF-beta pathway localized in different cellular compartments (Supplementary Fig. 6c
). This coordinate inhibition, as observed for other miRNAs49-51
, might confer robustness in this signaling network.
We unexpectedly identified a G-rich motif in most of the sequences associated with Ago2 regardless of the miRNA status in the cell. We believe this is a true biological association, rather than a technical artifact, based on the following observations. First, this motif is conserved above the general 3′UTR background even when matched for sequence content. Second, the genes containing this G-rich motif have significant overlap with the set of genes enriched in HA-tagged Ago2 immunoprecipitates from Dicer−/−
mESCs. Third, we only observe G bias in genic sequences, and not miRNA or intergenic sequences crosslinked to Ago2. Lastly, the enrichment of G residues is not likely due to CLIP itself as there are no described G biases in the literature for any of the steps involved (Supplementary Notes
for further discussion).
Yet it remains unclear whether crosslinking to this G-rich sequence is due to Ago2 itself or a binding partner of Ago2. Given that UV-crosslinking forms covalent bonds between protein and RNA that are within angstroms, a potential binding partner would have to be in close proximity to Ago2 and the mRNA target. Indeed, several proteins that co-immunoprecipitate with Ago2 have binding preference for G-rich sequences, including HNRNP-H and FMRP52-54
, although we only observed one Ago2-dependent RNAprotein complex close to the molecular weight of native Ago2 in the CLIP procedure. Alternatively, Ago2 itself could have a previously unidentified preference for binding G-rich sequences. In either case, when a G-rich sequence occurs near a miRNA binding site, it could give the Ago2/miRNA complexes a higher affinity for this region and thus lead to increased probability that the mRNA is targeted for degradation and/or inhibition of translation. In three cases examined, this G-rich motif modulates the level of miRNA-dependent regulation by the miR-290~295
-related seed motif, but imparts no regulation by itself. Therefore, identification of this association indicates the value of Ago2-CLIP data from Dicer−/−
mESCs as an invaluable background in delineating bona fide