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Updated: Mar 3, 2026

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs
Published on: June 12, 2018
A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis
Thomas Treiber1, Nora Treiber1, Uwe Plessmann2
1Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.
Abstract:
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
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