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. 2015 Jun 23;34(17):2237–2254. doi: 10.15252/embj.201490643

Figure 1.

Figure 1

Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening

  1. Setup of the RNAi screen in mouse primary cortical neurons. Neurons (5 days in vitro (DIV)) were co-transfected with the miR-134-responsive pGL3-UBE3A-3′UTR luciferase reporter (pGL3-UBE3A-luc) together with miR-134 duplex RNA and siRNA (one per condition) directed against 286 RBPs expressed in the mouse cortex (3 siRNAs per RBP). Luciferase activity was determined 3 days after transfection.
  2. Table of genes which are either known to be involved in the regulation of miRNA activity (“known genes”) or whose knockdown led to an at least 50% relief of miRNA-mediated repression for at least two out of the three siRNAs tested (“hits”). Values are the average of three replicate experiments and represent the percent relief of miRNA-mediated repression for the indicated siRNA compared to a condition without siRNA. Note that two known genes (Tnrc6c, Ddx6) were also present within the 12 hits.
  3. Luciferase reporter assay in primary rat cortical neurons transfected with pGL4-UBE3A-luc and the indicated siRNAs in the presence or absence of miR-134 duplex RNA. The ratio of normalized luciferase activity from neurons with (“+miR”) to neurons without co-transfected miR-134 (“−miR”) is plotted. Values are the average from three independent experiments ± standard deviation. *P < 0.05 (unpaired t-test compared to control siRNA).
  4. Luciferase reporter assay as in (C) with pGL4-APT1 3′UTR (APT1-luc) in the presence (“+miR”) or absence (“−miR”) of miR-138. n = 3. *P < 0.05 (unpaired t-test compared to control siRNA).