(A) The reporter constructs used in C and D. pFL-BRCA1 and pFL-BRCA1-mut have no introns, whereas pFL-BRCA1in and pFL-BRCA1in-mut each harbor two introns downstream of PTC. The miREs for miR-137 and miR-544 were mutated to synonymous codons in pFL-BRCA1-mut and pFL-BRCA1in-mut. (B) Identification of the surveillance mechanism pertinent to each reporter. (C) Left panel: miRNA-mediated surveillance and EJC-NMD are not mutually exclusive. Knockdown of RISC core component GW182 alleviated the repression of BRCA1 reporters, regardless of the presence of the downstream introns. The relative FL expression level represents the firefly/Renilla luciferase ratio in the presence of a control siRNA (siNC) versus an siRNA targeting GW182 (siGW182). Right panel: expression level of GW182 protein in HeLa-tTA cells treated with siNC or siGW182. Tubulin served as a loading control. (D) Additive effects of miRNA-mediated surveillance and EJC-NMD. Each reporter in A was co-transfected with miR-137, miR-544, or a control small RNA (siNC), together with a Renilla luciferase reporter into HEK293 cells. The relative FL expression level represents the firefly/Renilla luciferase ratio of each EJC-NMD- and/or miRNA-responsive reporter relative to pFL-BRCA1-mut. (E) Reporter constructs for miRE function validation of candidates identified from HCT-116 exome and RNA sequencing data. The PTC-STOP region of each candidate was fused to a firefly luciferase (FL) ORF in the same manner as for APC and BRCA1. A control Renilla luciferase (RL) reporter was expressed from a second promoter in the same construct (pRF-candidate). Each potential miRE identified from the screening was mutated to obtain a series of miRE mutant reporters (pRF-candidate-miREmut). (F–I) Experimental verification of functional miREs in the PTC-STOP region of selected candidates. The wild-type or miRE mutant version of each candidate reporter was co-transfected into HEK293 cells with a cognate miRNA mimic. The activity of each miRE (fold increase) was calculated from the normalized levels of firefly luciferase activity for pRF-candidate versus pRF-candidate-miREmut in the presence of the cognate miRNA mimic.