Systematic screens of proteins binding to synthetic microRNA precursors.
Towbin, H, Wenter, P, Guennewig, B, Imig, J, Zagalak, JA, Gerber, AP and Hall, J (2013) Systematic screens of proteins binding to synthetic microRNA precursors. Nucleic Acids Research, 41 (3), e47.
Nucl. Acids Res.-2013-Towbin-e47.pdf
Available under License : See the attached licence file.
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.
|Divisions :||Faculty of Health and Medical Sciences > School of Biosciences and Medicine > Department of Microbial and Cellular Sciences|
|Identification Number :||10.1093/nar/gks1197|
|Additional Information :||� The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited.|
|Depositing User :||Symplectic Elements|
|Date Deposited :||10 Jul 2013 10:23|
|Last Modified :||23 Sep 2013 20:09|
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