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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Towbin H,Wenter P,Guennewig B,Imig J,Zagalak JA,Gerber AP,Hall J

doi

10.1093/nar/gks1197

subject

Has Abstract

pub_date

2013-02-01 00:00:00

pages

e47

issue

3

eissn

0305-1048

issn

1362-4962

pii

gks1197

journal_volume

41

pub_type

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