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• W3033112679 abstract "•The authors describe a mouse strain harboring a Cre-regulated Halo-Ago2 knockin allele•The model streamlines the experimental identification of miRNA-mRNA interactions•The authors identify miRNA targets in mESCs, embryos, normal tissues, and tumors The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions. The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions. A key challenge in deciphering the biological functions of microRNAs (miRNAs) remains the identification of their targets in vivo under physiological and pathological conditions. Although significant progress has been made in computational methods to predict miRNA binding sites (Agarwal et al., 2015Agarwal V. Bell G.W. Nam J.-W. Bartel D.P. Predicting effective microRNA target sites in mammalian mRNAs.eLife. 2015; 4: e05005Crossref Scopus (3989) Google Scholar, Bartel, 2009Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15352) Google Scholar, Friedman et al., 2009Friedman R.C. Farh K.K. Burge C.B. Bartel D.P. Most mammalian mRNAs are conserved targets of microRNAs.Genome Res. 2009; 19: 92-105Crossref PubMed Scopus (6126) Google Scholar, Grimson et al., 2007Grimson A. Farh K.K. Johnston W.K. Garrett-Engele P. Lim L.P. Bartel D.P. MicroRNA targeting specificity in mammals: determinants beyond seed pairing.Mol. Cell. 2007; 27: 91-105Abstract Full Text Full Text PDF PubMed Scopus (2970) Google Scholar), these methods do not take into account several known and unknown variables that determine whether a “potential” target site is in fact available and bound by a miRNA in a given cellular context. To complement computational approaches, biochemical methods to purify Ago2-miRNA-mRNA complexes have been developed (Chi et al., 2009Chi S.W. Zang J.B. Mele A. Darnell R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature. 2009; 460: 479-486Crossref PubMed Scopus (1377) Google Scholar, Grosswendt et al., 2014Grosswendt S. Filipchyk A. Manzano M. Klironomos F. Schilling M. Herzog M. Gottwein E. Rajewsky N. Unambiguous identification of miRNA:target site interactions by different types of ligation reactions.Mol. Cell. 2014; 54: 1042-1054Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, Hafner et al., 2010Hafner M. Landthaler M. Burger L. Khorshid M. Hausser J. Berninger P. Rothballer A. Ascano Jr., M. Jungkamp A.C. Munschauer M. et al.Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.Cell. 2010; 141: 129-141Abstract Full Text Full Text PDF PubMed Scopus (2045) Google Scholar, Helwak et al., 2013Helwak A. Kudla G. Dudnakova T. Tollervey D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding.Cell. 2013; 153: 654-665Abstract Full Text Full Text PDF PubMed Scopus (858) Google Scholar, König et al., 2010König J. Zarnack K. Rot G. Curk T. Kayikci M. Zupan B. Turner D.J. Luscombe N.M. Ule J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.Nat. Struct. Mol. Biol. 2010; 17: 909-915Crossref PubMed Scopus (728) Google Scholar, Moore et al., 2015Moore M.J. Scheel T.K. Luna J.M. Park C.Y. Fak J.J. Nishiuchi E. Rice C.M. Darnell R.B. miRNA-target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity.Nat. Commun. 2015; 6: 8864Crossref PubMed Scopus (172) Google Scholar, Van Nostrand et al., 2016Van Nostrand E.L. Pratt G.A. Shishkin A.A. Gelboin-Burkhart C. Fang M.Y. Sundararaman B. Blue S.M. Nguyen T.B. Surka C. Elkins K. et al.Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).Nat. Methods. 2016; 13: 508-514Crossref PubMed Scopus (575) Google Scholar). Although the details vary, these methods rely on the use of antibodies to precipitate Argonaute-containing complexes, usually after UV crosslinking, followed by high-throughput sequencing of the associated mRNAs. While these methods have been applied with substantial success to map miRNA-mRNA interactions in cell lines, they are used much less extensively in vivo due to their technical complexity and the lack of efficient ways to restrict the analysis to specific cell types within a tissue. To overcome these limitations, we have developed a method, Halo-enhanced Ago2 pull-down (HEAP), which utilizes a tagged version of the Ago2 protein and allows the direct purification of Ago2-containing complexes bypassing the need for radiolabeling, immunoprecipitation, and gel purification. To facilitate the application of this method in vivo, we have generated a mouse strain in which a conditional allele of Halo-tagged Ago2 is knocked into the endogenous Ago2 locus and activated upon exposure to Cre recombinase. To benchmark the HEAP method, we applied it to identify miRNA targets in diverse cellular contexts, including murine embryonic stem cells (mESCs), wild-type and miR-17∼92 null mid-gestation mouse embryos, adult mouse lungs, adult mouse brains, and three distinct autochthonous mouse models of human lung and brain cancers. As a result, we have identified a large number of miRNA targets at high resolution and demonstrated the reproducibility and sensitivity of the HEAP method. The datasets and the tools generated in this study reveal the complex landscape of miRNA targeting in vivo and will facilitate future studies aimed at characterizing the biological functions of this important class of small non-coding RNAs under physiological and pathological conditions. The HaloTag is a 33-kDa haloalkane dehalogenase encoded by the DhaA gene from Rhodococcus rhodochrous that has been mutagenized to form an irreversible covalent bond to synthetic chloroalkane ligands (collectively known as HaloTag ligands) (Encell et al., 2012Encell L.P. Friedman Ohana R. Zimmerman K. Otto P. Vidugiris G. Wood M.G. Los G.V. McDougall M.G. Zimprich C. Karassina N. et al.Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.Curr. Chem. Genomics. 2012; 6: 55-71Crossref PubMed Google Scholar, Los et al., 2008Los G.V. Encell L.P. McDougall M.G. Hartzell D.D. Karassina N. Zimprich C. Wood M.G. Learish R. Ohana R.F. Urh M. et al.HaloTag: a novel protein labeling technology for cell imaging and protein analysis.ACS Chem. Biol. 2008; 3: 373-382Crossref PubMed Scopus (1294) Google Scholar). Linking the chloroalkane ligand to a solid substrate enables the efficient purification of fusion proteins containing the HaloTag (Figure 1A). Importantly, Gu and colleagues have recently used the HaloTag together with UV crosslinking to efficiently identify RNA targets of the RNA binding protein PTB (Gu et al., 2018Gu J. Wang M. Yang Y. Qiu D. Zhang Y. Ma J. Zhou Y. Hannon G.J. Yu Y. GoldCLIP: gel-omitted ligation-dependent CLIP.Genomics Proteomics Bioinformatics. 2018; 16: 136-143Crossref PubMed Scopus (15) Google Scholar). To determine whether a similar strategy can be employed to purify complexes containing Ago2 proteins bound to miRNA and target mRNAs, we fused the HaloTag to the N terminus of Ago2 (Halo-Ago2; Figure 1A). When expressed in Ago2−/− mouse embryonic fibroblasts (MEFs) (O’Carroll et al., 2007O’Carroll D. Mecklenbrauker I. Das P.P. Santana A. Koenig U. Enright A.J. Miska E.A. Tarakhovsky A. A Slicer-independent role for Argonaute 2 in hematopoiesis and the microRNA pathway.Genes Dev. 2007; 21: 1999-2004Crossref PubMed Scopus (293) Google Scholar), the Halo-Ago2 fusion protein localized largely to the cytoplasm, while the HaloTag alone displayed uniform localization to both the cytoplasm and the nucleus (Figure 1B; Data S1). Importantly, the Halo-Ago2 construct was nearly as effective as wild-type Ago2 at rescuing RNAi in Ago2−/− MEFs, indicating that the Halo-Ago2 fusion protein retains slicing activity (Figure 1C). To avoid artifacts due to ectopic expression of Halo-Ago2 and enable the isolation of Ago2 complexes directly from murine tissues, we knocked in the HaloTag cassette into the endogenous Ago2 locus in mESCs (Figure 1D). In this knockin allele, the HaloTag is separated from the first coding exon of Ago2 by an in-frame loxP-STOP-IRES-FLAG-loxP (LSL) cassette (Ago2Halo-LSL). Cells harboring this allele express a bicistronic mRNA encoding for two proteins, the HaloTag and a Flag-Ago2 fusion protein whose translation is initiated by an internal ribosomal entry site (IRES). Upon expression of the Cre recombinase, the LSL cassette is excised and the HaloTag is now brought in frame with the first coding exon of Ago2, thus resulting in expression of the Halo-Ago2 fusion protein (Figures 1D and 1E). The recombined allele expressing the Halo-Ago2 fusion will be hereafter referred to as Ago2Halo. We first tested whether the Ago2Halo allele could be used to map miRNA-mRNA interactions in mESCs. For these experiments, we adapted the Ago2 high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (Ago2 HITS-CLIP) method originally developed by the Darnell group (Chi et al., 2009Chi S.W. Zang J.B. Mele A. Darnell R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature. 2009; 460: 479-486Crossref PubMed Scopus (1377) Google Scholar) with two significant streamlining modifications enabled by the covalent bond between Halo-Ago2 and the HaloTag ligand. First, instead of using anti-Ago2 antibodies to isolate Ago2-containing complexes, we used Sepharose beads covalently linked to the HaloTag ligand. Second, the radiolabeling and SDS-PAGE purification step necessary in CLIP protocols to purify RNAs bound to Ago2 were omitted and replaced by extensive washes followed by direct RNA extraction from beads, library construction, and high-throughput sequencing of Halo-Ago2-bound miRNAs and mRNAs. We refer to this method as HEAP (Figure 1F). By performing HEAP, two types of libraries are generated: a target library (mRNAs) and a miRNA library (Figures 1F and S1A). The former allows the identification of miRNA binding sites on their targets, while the latter provides an estimate of miRNA abundance. When mapped to the mouse genome, HEAP mRNA libraries generated from Ago2Halo/+ mESCs, but not those generated from control Ago2Halo-LSL/+ cells, produced well-defined “clusters” of reads, hereafter referred to as “peaks” (Figures S1A and S1B). To facilitate the identification of these peaks, we adapted the “SMInput” protocol used in enhanced-CLIP (eCLIP) (Van Nostrand et al., 2016Van Nostrand E.L. Pratt G.A. Shishkin A.A. Gelboin-Burkhart C. Fang M.Y. Sundararaman B. Blue S.M. Nguyen T.B. Surka C. Elkins K. et al.Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).Nat. Methods. 2016; 13: 508-514Crossref PubMed Scopus (575) Google Scholar) and generated input control libraries from size-matched RNA fragments isolated after the limited RNase protection step (Figure 1F). We first identified putative peaks using the CLIPanalyze package (https://bitbucket.org/leslielab/clipanalyze), an improved peak-calling algorithm based on edge detection technique similar to methods from image processing (Hsin et al., 2018Hsin J.P. Lu Y. Loeb G.B. Leslie C.S. Rudensky A.Y. The effect of cellular context on miR-155-mediated gene regulation in four major immune cell types.Nat. Immunol. 2018; 19: 1137-1145Crossref PubMed Scopus (64) Google Scholar, Lianoglou et al., 2013Lianoglou S. Garg V. Yang J.L. Leslie C.S. Mayr C. Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression.Genes Dev. 2013; 27: 2380-2396Crossref PubMed Scopus (240) Google Scholar, Loeb et al., 2012Loeb G.B. Khan A.A. Canner D. Hiatt J.B. Shendure J. Darnell R.B. Leslie C.S. Rudensky A.Y. Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting.Mol. Cell. 2012; 48: 760-770Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). CLIPanalyze uses the input control libraries as background to assign a p value to each peak, performing library size normalization based on reads aligned across the genome outside of putative peaks (see also STAR Methods for additional details). To determine the sensitivity and reproducibility of the HEAP method, we generated HEAP libraries from three Ago2Halo/+ mESC clones (using 1.5 × 108 cells per library). By combining the three libraries, CLIPanalyze identified a total of 30,564 putative Ago2 binding sites at an adjusted p value cutoff of 0.05. Previous studies have demonstrated that 3′ untranslated regions (3′ UTRs) of mRNAs are the preferred, although not exclusive, sites of interaction between miRNAs and mRNAs (Bartel, 2018Bartel D.P. Metazoan MicroRNAs.Cell. 2018; 173: 20-51Abstract Full Text Full Text PDF PubMed Scopus (1529) Google Scholar, Chi et al., 2009Chi S.W. Zang J.B. Mele A. Darnell R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature. 2009; 460: 479-486Crossref PubMed Scopus (1377) Google Scholar, Sarshad et al., 2018Sarshad A.A. Juan A.H. Muler A.I.C. Anastasakis D.G. Wang X. Genzor P. Feng X. Tsai P.F. Sun H.W. Haase A.D. et al.Argonaute-miRNA complexes silence target mRNAs in the nucleus of mammalian stem cells.Mol. Cell. 2018; 71: 1040-1050.e8Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Consistent with these findings, the majority of HEAP peaks we identified in mESCs mapped to 3′ UTRs, followed by sites mapping to protein coding sequences (CDS) (Figures 2A and S1C). The fractions of 3′ UTR and CDS peaks increased monotonically with their statistical significance, while intergenic and intronic peaks had the opposite behavior. For example, when examining the 1,000 most statistically significant peaks, greater than 50% of them mapped to 3′ UTRs and less than 3% mapped to introns (Figure 2A). To measure reproducibility, we applied the CLIPanalyze algorithm independently to each library and performed pairwise irreproducible discovery rate (IDR) (Li et al., 2011Li Q. Brown J.B. Huang H. Bickel P.J. Measuring reproducibility of high-throughput experiments.Ann. Appl. Stat. 2011; 5: 1752-1779Crossref Scopus (529) Google Scholar) analysis. On average, this analysis identified 80% of peaks as reproducible at IDR < 0.05, demonstrating the robustness of the HEAP method (Figure S1D). We also generated a series of HEAP libraries using decreasing numbers of mESCs (from 1.5 × 108 to 1 × 103). As expected, the total number of confidently identified peaks progressively decreased as the amount of starting material was reduced (Figure S1E). The most robust peaks could be identified in libraries generated from as few as 5 × 105 mESCs (Figure S1F), but for optimal results, we recommend starting from a minimum of 1 × 107 mESCs. Since mESCs have little cytoplasm, the detection limit is likely to be lower for cell types with more abundant cytoplasm. To gain additional insights into the nature of peaks identified by HEAP, we searched for enriched 7-mers in the sequences underlying peaks mapping to 3′ UTRs (Figure S1G). Inspection of the resulting motifs revealed a marked enrichment for seed matches corresponding to miRNA families whose members are collectively highly expressed in mESCs (Figures 2B and S1H). We also observed a positive correlation between the relative abundance of individual miRNA families (estimated from the miRNA libraries) and the number of corresponding peaks identified by HEAP (Figure 2C). To directly test whether the peaks identified by HEAP reflect true miRNA-mRNA interactions, we selected a robust peak identified in the 3′ UTR of the Lefty2 mRNA (Figure 2D). The sequence underlying this peak includes a highly conserved 8-mer that is complementary to the miR-291-3p seed (Figure S1I). We used CRISPR-Cas9 and homologous recombination in mESCs to introduce point mutations designed to disrupt this seed match (Figure S1I). HEAP libraries generated from two independent Lefty2MUT clones showed complete and selective loss of the Lefty2 peak, further demonstrating the ability of the HEAP method to map bona fide miRNA-mRNA interactions in cells (Figures 2D and 2E). To assess the ability of HEAP to identify functional miRNA binding sites, we analyzed an RNA sequencing (RNA-seq) dataset generated by Bosson and colleagues from mESCs null for all four Argonaute proteins (Ago1-4−/−) in the presence or absence of exogenously expressed FLAG- and hemagglutinin (HA)-tagged AGO2 (FHAGO2; Bosson et al., 2014Bosson A.D. Zamudio J.R. Sharp P.A. Endogenous miRNA and target concentrations determine susceptibility to potential ceRNA competition.Mol. Cell. 2014; 56: 347-359Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar; GEO: GSE61348). Introduction of FHAGO2 in Ago1-4−/− cells should restore miRNA function, causing repression of their targets. In agreement with this prediction, miRNA targets identified by HEAP were preferentially repressed upon FHAGO2 reintroduction (Figure S2A). The effect was particularly strong for targets assigned by HEAP to the most abundantly expressed miRNA families in mESCs. For example, we observed the strongest repression for targets of the miR-291-3p, miR-17-5p and miR-148-3p families, three miRNA families that account for greater than 12% of all miRNAs in mESCs (Figure S2A; data not shown). Peaks with lower adjusted p values or higher log2-fold changes (HEAP versus input control) were associated with stronger target repression (Figure S2B). As expected, peaks mapping to 3′ UTRs were associated with strongest target repression compared to peaks mapping to other genomic annotations (Figure S2C). This analysis also allowed us to compare miRNA targets identified by HEAP to those previously identified by Bosson et al. in Ago1-4−/−-FHAGO2 mESCs using individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), a well-established variant of HITS-CLIP (König et al., 2010König J. Zarnack K. Rot G. Curk T. Kayikci M. Zupan B. Turner D.J. Luscombe N.M. Ule J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.Nat. Struct. Mol. Biol. 2010; 17: 909-915Crossref PubMed Scopus (728) Google Scholar). By applying the CLIPanalyze peak calling algorithm, we identified 6,813 miRNA binding sites in their iCLIP library and nearly twice as many (on average 13,532) in each of the three HEAP mESC libraries. The iCLIP library also identified fewer peaks mapping to 3′ UTR and more peaks mapping to intergenic regions compared to the HEAP libraries (Figure S2D). 3′ UTR targets for miR-291-3p seed family identified by both methods were associated with strong repression of the corresponding genes upon FHAGO2 reintroduction (Figure 2F). The overlap between miR-291-3p binding sites identified by iCLIP and HEAP in 3′ UTRs was partial, with the HEAP target pool being nearly twice as large (Figure S2E). Importantly, the targets identified only by HEAP also displayed strong repression upon FHAGO2 reintroduction, indicating that they are functional miRNA binding sites (Figure 2F). We further confirmed the ability of HEAP to identify functional miRNA targets by measuring mRNA and protein expression changes of HEAP targets upon inactivation of Dicer1, the key enzyme responsible for miRNA maturation, in mESCs (Figure S2F). Collectively, these results show that HEAP provides an effective method to identify miRNA-mRNA interactions in cells. The accurate identification of miRNA targets in vivo and in a cell-type-specific context is essential to dissect the functions of miRNAs in development, homeostasis, and disease. To translate the HEAP method to an in vivo setting, we used mESCs harboring the Cre-inducible Halo-Ago2 allele to generate Ago2Halo-LSL/+ mice. We then crossed these animals to CAG-Cre mice (Sakai and Miyazaki, 1997Sakai K. Miyazaki J.-i. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission.Biochem. Biophys. Res. Commun. 1997; 237: 318-324Crossref PubMed Scopus (422) Google Scholar) to delete the LSL cassette and induce ubiquitous expression of the endogenous Halo-Ago2 allele. PCR in MEFs and immunoblot analysis in MEFs and tissues derived from these mice confirmed efficient deletion of the LSL cassette and expression of the Halo-Ago2 protein (Figures 3A, S3A, and S3B). Although Ago2Halo/+ and Ago2Halo-LSL/+ mice were obtained at the expected Mendelian frequency and phenotypically indistinguishable from wild-type mice, homozygous mice for the Ago2Halo or the Ago2Halo-LSL alleles were recovered at sub-Mendelian frequencies (9.9% and 11.9%, respectively, compared to the expected 25%; Figure 3B). The sub-Mendelian recovery of homozygous mice might reflect lower Ago2 expression levels compared to wild-type mice (Figures 3A and S3B) and/or an impaired miRNA-induced silencing complex (miRISC) formation or activity caused by the presence of the N-terminal tag. Size-exclusion chromatography in Ago2Halo/+ cells showed the Halo-Ago2 fusion protein co-eluting with wild-type Ago2 in high-molecular-weight complexes (Figure S3C), and pull-down experiments confirmed the physical interaction between Halo-Ago2 and Tnrc6a, a core component of the miRISC (Figure S3D). Furthermore, reporter experiments using multiple luciferase reporter constructs harboring well-characterized miRNA binding sites, as well as a highly sensitive two-color fluorescent reporter system (Mukherji et al., 2011Mukherji S. Ebert M.S. Zheng G.X.Y. Tsang J.S. Sharp P.A. van Oudenaarden A. MicroRNAs can generate thresholds in target gene expression.Nat. Genet. 2011; 43: 854-859Crossref PubMed Scopus (477) Google Scholar), showed no detectable differences in miRNA-mediated repression between wild-type and Ago2Halo/Halo MEFs (Figures S3E and S3F). A careful comparison of RNA-seq libraries generated from wild-type and Ago2Halo/Halo cells, however, revealed a slight preferential de-repression of targets of the most highly expressed miRNA families (Figure S3G). Due to the importance of miRNA-mediated gene regulation during embryonic development, it is possible that this modest perturbation of miRISC activity is responsible for the observed reduced viability of homozygous mice. To test whether endogenously expressed Halo-Ago2 can be used to identify miRNA targets in vivo, we crossed Ago2Halo/+ mice to mice harboring a targeted deletion of the miR-17∼92 locus (Mirc1), a polycistronic miRNA cluster encoding six distinct miRNAs, which has been shown to be essential for mammalian development (Han et al., 2015Han Y.C. Vidigal J.A. Mu P. Yao E. Singh I. González A.J. Concepcion C.P. Bonetti C. Ogrodowski P. Carver B. et al.An allelic series of miR-17 ∼ 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron.Nat. Genet. 2015; 47: 766-775Crossref PubMed Scopus (78) Google Scholar, Ventura et al., 2008Ventura A. Young A.G. Winslow M.M. Lintault L. Meissner A. Erkeland S.J. Newman J. Bronson R.T. Crowley D. Stone J.R. et al.Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters.Cell. 2008; 132: 875-886Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). We generated HEAP libraries from Ago2Halo/+; miR-17∼92+/+ (miR-17∼92-WT), Ago2Halo/+; miR-17∼92+/− (miR-17∼92-HET) and Ago2Halo/+; miR-17∼92−/− (miR-17∼92-KO) embryonic day 13.5 (E13.5) embryos (Figure 3C). At an adjusted p value cutoff of 0.01, HEAP identified a total of 8,661 peaks in these libraries, with a distribution across genomic annotations similar to that observed in mESCs (Figure S4A). Importantly, the intensity of peaks containing seed matches to members of the miR-17∼92 cluster was markedly reduced—in a dose-dependent fashion—in the libraries generated from miR-17∼92-HET and miR-17∼92-KO embryos (Figure 3D). The murine genome contains two additional miRNA clusters that are paralogs to miR-17∼92 and encode similar miRNAs (Ventura et al., 2008Ventura A. Young A.G. Winslow M.M. Lintault L. Meissner A. Erkeland S.J. Newman J. Bronson R.T. Crowley D. Stone J.R. et al.Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters.Cell. 2008; 132: 875-886Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar), which may explain some residual Halo-Ago2 binding to these sites even in the homozygous mutants. Using an RNA-seq dataset previously generated in the lab from E9.5 embryos harboring an allelic series of miR-17∼92 mutant alleles (Han et al., 2015Han Y.C. Vidigal J.A. Mu P. Yao E. Singh I. González A.J. Concepcion C.P. Bonetti C. Ogrodowski P. Carver B. et al.An allelic series of miR-17 ∼ 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron.Nat. Genet. 2015; 47: 766-775Crossref PubMed Scopus (78) Google Scholar; GEO: GSE63813), we demonstrated that HEAP targets containing seed matches for miR-17/20-5p, miR-19-3p, and miR-92-3p mediated strong target repression (Figure S4B). The effect was particularly evident when considering genes harboring HEAP peaks for miR-17/20-5p and miR-92-3p, whose signal intensities were reduced in the miR-17∼92-KO embryo, confirming the importance of combining biochemical and genetic approaches to study miRNA function. Interestingly, we also identified a sizeable fraction of reproducible peaks (4%) mapping to non-coding RNAs. These included two previously uncharacterized miR-17∼92-dependent sites matching the miR-92-3p seed in the long non-coding RNA Cyrano (Kleaveland et al., 2018Kleaveland B. Shi C.Y. Stefano J. Bartel D.P. A network of noncoding regulatory RNAs acts in the mammalian brain.Cell. 2018; 174: 350-362.e17Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, Ulitsky et al., 2011Ulitsky I. Shkumatava A. Jan C.H. Sive H. Bartel D.P. Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution.Cell. 2011; 147: 1537-1550Abstract Full Text Full Text PDF PubMed Scopus (846) Google Scholar) (Figure 3E). Importantly, we observed significant upregulation of Cyrano in mouse E9.5 embryos lacking miR-92a-1, but not in mice harboring selective deletion of the other members of the cluster (Figure 3F) (Han et al., 2015Han Y.C. Vidigal J.A. Mu P. Yao E. Singh I. González A.J. Concepcion C.P. Bonetti C. Ogrodowski P. Carver B. et al.An allelic series of miR-17 ∼ 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron.Nat. Genet. 2015; 47: 766-775Crossref PubMed Scopus (78) Google Scholar), suggesting these binding sites are functional. These results demonstrate the usefulness of the Halo-Ago2 mouse strain in facilitating the identification of miRNA targets in vivo. To directly compare the performance of HEAP to immunoprecipitation-based approaches in vivo, we next generated libraries from the cortex of P13 Ago2Halo/+ mice, a tissue from which high-quality miRNA target libraries have been previously generated by HITS-CLIP and CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP (Chi et al., 2009Chi S.W. Zang J.B. Mele A. Darnell R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature. 2009; 460: 479-486Crossref PubMed Scopus (1377) Google Scholar, Moore et al., 2015Moore M.J. Scheel T.K. Luna J.M. Park C.Y. Fak J.J. Nishiuchi E. Rice C.M. Darnell R.B. miRNA-target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity.Nat. Commun. 2015; 6: 8864Crossref PubMed Scopus (172) Google Scholar). Two HEAP libraries generated from the cortices of AgoHalo/+ mice produced 7,069 peaks at an adjusted p value cutoff of 0.05. This number of miRNA-mRNA interaction sites is comparable to that identified by Moore and colleagues (CLEAR-CLIP, GEO: GSE73059, n = 7,927) using 12 biological replicates (Figure" @default.
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• W3033112679 date "2020-07-01" @default.
• W3033112679 modified "2023-10-10" @default.
• W3033112679 title "High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down" @default.
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