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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Towbin H,Wenter P,Guennewig B,Imig J,Zagalak JA,Gerber AP,Hall Jdoi
10.1093/nar/gks1197subject
Has Abstractpub_date
2013-02-01 00:00:00pages
e47issue
3eissn
0305-1048issn
1362-4962pii
gks1197journal_volume
41pub_type
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