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. 2018 Oct 17;8:15316. doi: 10.1038/s41598-018-33596-4

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Luciferase reporter assays of candidate sites reveal antagonistic AGO2-PUM interactions. (A) A known Pumilio-regulated site from the 3′UTR of the Drosophila hunchback mRNA shows loss of regulation in PUM double knockout cells. (B) A strong AGO2 site in the LRIG3 3′UTR with a predicted miRNA seed shows loss of repression in DICER knockout, but not PUM double knockout, cells. (C) A subset of candidates show miRNA-dependent activity (PMEPA1, ZNF367, SNAPC1) or both miRNA and PUM dependence (CDKN1B-2, ATP6V1G1, FNIP1, RRAGD, TOB1, VLDLR). Candidate sites were tested in three conditions: wildtype T-REx-293, DICER knockout cells, and PUM double knockout cells. (D) Sites co-regulated by AGO and PUM sites show dependence on specific miRNAs in PUM double knockout cells. For FNIP1 and VLDLR, WT and mutant AGO peak constructs were used. For RRAGD and TOB1, WT and mutant miRNA seed constructs were used.