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. 2016 Feb 5;6:20109. doi: 10.1038/srep20109

Figure 2. Secondary screen for regulators of Or47b expression using gene-specific RNAi.

Figure 2

(A) Tabular summary of the target predictions made by three different algorithms for each miRNA whose over-expression reduces Or47b expression. (B) The number of genes that appear on the TargetScan candidate target lists in A. (C) Twenty-two genes, including Atf3, appear on the candidate target lists for 3 different miRNAs. (D) A schematized view of the Atf3 (A3-3) 3′-UTR showing the locations of seed matches for miR-2a and miR-33, which reduce Or47b expression. (E) Atf3 knockdown in all olfactory neurons using Peb-GAL4 reduces expression of an Or47b reporter, but spares expression of an Or92a reporter. (F) Antennal in situ hybridization with mixed riboprobes that recognize Or47b (blue) and Or92a (red) reveals that Atf3 knockdown using two different RNAi lines eliminates Or47b expression, but spares Or92a expression. (G) Over-expression of miR-2a-2 in olfactory neurons using Peb-GAL4 reduces the expression of Atf3 in the antenna. n = 2 biological replicates of 3 technical replicates each. ***p = 0.0004. (H) An Atf3::eGFP fusion protein lacking the Atf3 3′-UTR, which contains the miR-2a-2 binding site, rescues the loss of Or47b expression observed with miR-2a-2 over-expression.