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This research was supported by a Human Frontier Science Program Long Term Fellowship to Y.K. and by NIH grants GM0720777, NS056070, and a URF grant from the University or college of Pennsylvania to Z.M. Footnotes Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1133808. Recommendations Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281C297. [PubMed] [Google Scholar]Beitzinger, M., Peters, L., Zhu, J.Y., Kremmer, E., Meister, G. Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol. 2007;4:76C84. [PubMed] [Google Scholar]Carmell, M.A., Xuan, Z., Zhang, M.Q., Hannon, G.J. The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes & Dev. 2002;16:2733C2742. [PubMed] [Google Scholar]Easow, G., Teleman, A.A., Cohen, S.M. 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When the reporter mRNAs were used, reactions were digested with 30 models of RNase T1 (Roche) for 20 min at 37C. Cross-linked proteins were separated by NuPAGE (NuPAGE 4%C12% Bis-Tris, Invitrogen) and detected by storage-phosphor autoradiography. ACKNOWLEDGMENTS We thank users of our laboratory for stimulating discussions. This research was supported by a Naproxen Human Frontier Science Program Long Term Fellowship to Y.K. and by NIH grants GM0720777, NS056070, and a URF grant from the University or college of Pennsylvania to Z.M. Footnotes Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1133808. Recommendations Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281C297. [PubMed] [Google Scholar]Beitzinger, M., Peters, L., Zhu, J.Y., Kremmer, E., Meister, G. Identification of human microRNA targets from isolated argonaute protein complexes. RNA Biol. 2007;4:76C84. 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Crosslinking was performed for 30 min on ice by irradiation with a 365-nm hand-held lamp (EL series UV lamp, UVP). When the reporter mRNAs were used, reactions were digested with 30 units of RNase T1 (Roche) for 20 min at 37C. Cross-linked proteins were separated by NuPAGE (NuPAGE 4%C12% Bis-Tris, Invitrogen) and detected by storage-phosphor autoradiography. ACKNOWLEDGMENTS We thank members of our laboratory for stimulating discussions. This research was supported by a Human Frontier Science Program Long Term Fellowship to Y.K. and by NIH grants GM0720777, NS056070, and a URF grant from the University of Pennsylvania to Z.M. Footnotes Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1133808. REFERENCES Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281C297. [PubMed] [Google Scholar]Beitzinger, M., Peters, L., Zhu, J.Y., Kremmer, E., Meister, G. 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MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science. 2007;317:1764C1767. [PubMed] [Google Scholar]Meister, G., Landthaler, M., Dorsett, Y., Tuschl, T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA. 2004;10:544C550. [PMC free article] [PubMed] [Google Scholar]Mili, S., Steitz, J.A. Evidence for reassociation of RNA-binding proteins after cell lysis: Implications for the interpretation of immunoprecipitation analyses. RNA. 2004;10:1692C1694. [PMC free article] [PubMed] [Google Scholar]Moore, M.J., Query, C.C. Uses of site-specifically modified RNAs constructed by RNA ligation. In: Smith C., editor. RNACprotein interactions: A practical approach. Oxford University Press; Oxford,.2007;317:1764C1767. of total HeLa lysate, or beads or supernatant in the lysis buffer obtained from 2A8 IP. Where indicated, the lysate or the beads were pre-incubated for 30 min at 28C with 25 pmol of let-7b inhibitor (miRIDIAN MicroRNA Inhibitor, Dharmacon) before the incubation with the labeled RNAs. Crosslinking was performed for 30 min on ice by irradiation with a 365-nm hand-held lamp (EL series UV lamp, UVP). When the reporter mRNAs were used, reactions were digested with 30 units of RNase T1 (Roche) for 20 min at 37C. Cross-linked proteins were separated by NuPAGE (NuPAGE 4%C12% Bis-Tris, Invitrogen) and detected by storage-phosphor autoradiography. ACKNOWLEDGMENTS We thank members of our laboratory for stimulating discussions. This research was supported by a Human Frontier Science Program Long Term Fellowship to Y.K. and by NIH grants GM0720777, NS056070, and a URF grant from the University of Pennsylvania to Z.M. Footnotes Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1133808. REFERENCES Bartel, D.P. 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Oxford College or university Press; Oxford, UK: 1998. pp. 75C108. [Google Scholar]Moore, M.J., Clear, P.A. Site-specific changes of pre-mRNA: The 2-hydroxyl organizations in the splice sites. Technology. 1992;256:992C997. [PubMed] [Google.Genes & Dev. using the 2A8 anti-Ago monoclonal antibody (Nelson et al. 2007), or non-immune mouse serum as adverse control. Crosslinking 32P-tagged RNA (10,000 cpm) had been incubated for 70 min at 28C in 10 L of total HeLa lysate, or beads or supernatant in the lysis buffer from 2A8 IP. Where indicated, the lysate or the beads had been pre-incubated for 30 min at 28C with 25 pmol of allow-7b inhibitor (miRIDIAN MicroRNA Inhibitor, Dharmacon) prior to the incubation using the tagged RNAs. Crosslinking was performed for 30 min on snow by irradiation having a 365-nm hand-held light (Un series UV light, UVP). When the reporter mRNAs had been used, reactions had been digested with 30 devices of RNase T1 (Roche) for 20 min at 37C. Cross-linked protein had been separated by NuPAGE (NuPAGE 4%C12% Bis-Tris, Invitrogen) and recognized by storage-phosphor autoradiography. ACKNOWLEDGMENTS We say thanks to people of our lab for stimulating conversations. This study was supported with a Human being Frontier Technology Program LONG-TERM Fellowship to Y.K. and by NIH grants or loans GM0720777, NS056070, and a URF give from the College or university of Pa to Z.M. Footnotes Content published online before print. Content and publication day are in http://www.rnajournal.org/cgi/doi/10.1261/rna.1133808. Referrals Bartel, D.P. MicroRNAs: Genomics, biogenesis, system, and function. Cell. 2004;116:281C297. [PubMed] [Google Scholar]Beitzinger, M., Peters, L., Zhu, J.Con., Kremmer, E., Meister, G. Recognition of human being microRNA focuses on from isolated argonaute proteins complexes. RNA Biol. 2007;4:76C84. [PubMed] [Google Scholar]Carmell, M.A., Xuan, Z., Zhang, M.Q., Hannon, G.J. 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