Lin28a Uses Distinct Mechanisms of Binding to RNA and Affects miRNA Levels Positively and Negatively

Nowak JS, et al., 23(3):317-332, RNA, 2017

Cell lineage abnormal 28a (Lin28a) is an essential RNA-binding protein, which plays a key role in the regulation of biogenesis of miRNAs from the let-7 family. Reported herein are the molecular and biophysical data to demonstrate that Lin28a uses two distinct mechanisms of binding to pre-let-7a and pre-miRNA-9. Binding affinity of recombinant Lin28 to pre-miR-9 (wild-type and mutant) and pre-let-7-a (wild-type and mutant) were studied by Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Streptavidin Biosensor probes was used to perform all BLI experiments. Biosensor tips were immobilized with biotinylated pre-miRNA-9 or pre-let-7a-1 RNAs and immersed in a concentration series of Lin28a. Equilibrium binding isotherms were obtained by plotting the signal intensity (nm shift) against analyte concentration (μM). The KD values were determined by fitting the curve using non-linear regression. Results obtained by the BLI were validated using electrophoretic mobility shift assays (EMSA). Overall findings provide a better understanding of the roles played by Lin28a in terms of differentiating the cells and the regulation of RNA processing in cells.

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