title: Systematic screens of proteins binding to synthetic microRNA precursors.
abstract: We describe a new, broadly applicable methodology for screening in parallel interactions of RNA-binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre-miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts. Detection of bound RBPs is achieved by protein or tag-specific antibodies allowing crude cell lysates to be used as a source of RBP. We selected 70 pre-miRNAs with phylogenetically conserved loop regions and 25 precursors of other well-characterized miRNAs for chemical synthesis in 3'-biotinylated form. An equivalent set in unmodified form served as inhibitors in affinity determinations. By testing three RBPs known to regulate miRNA biogenesis on this set of pre-miRNAs, we demonstrate that Lin28 and hnRNP A1 from cell lysates or as recombinant protein domains recognize preferentially precursors of the let-7 family, and that KSRP binds strongly to pre-miR-1-2.
authors.affiliation: Institute of Pharmaceutical Sciences, ETH Zürich, CH-8093 Zürich, Switzerland.
authors.forename: Harry
authors.initials: H
authors.lastname: Towbin
authors.forename: Philipp
authors.initials: P
authors.lastname: Wenter
authors.forename: Boris
authors.initials: B
authors.lastname: Guennewig
authors.forename: Jochen
authors.initials: J
authors.lastname: Imig
authors.forename: Julian A
authors.initials: JA
authors.lastname: Zagalak
authors.forename: André P
authors.initials: AP
authors.lastname: Gerber
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hall
mesh_terms: D057809:HEK293 Cells; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000138:chemical synthesis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C105789:KHSRP protein, human; C000715187:Lin28A protein, human; D035683:MicroRNAs; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D015534:Trans-Activators