Fig. 2: TDMD triggers preferably locate in the noncoding region of RNA transcripts.

a Illustration of the inducible sfGFP reporter system containing an engineered trigger against miR-16. b Base-pairing pattern of miR-16 and its trigger, which mimics the pattern of CYRANO: miR-7. miR-16 seed region is highlighted in red. c Northern blot analyses of miR-16 in cells expressing inducible sfGFP reporters with the CDS or 3ʹ UTR trigger against miR-16. Cells were treated with different doses of Dox indicated above the blots. The normalized miRNA abundance and standard deviation are shown below each miRNA. miRNA abundance is normalized to U6. The miRNA abundance in CDS and 3ʹ UTR without Dox (0) treatment is normalized to 1, respectively. d The quantification of northern blots in c. miR-16 levels are normalized to U6 RNA and the value in cells without Dox treatment is set to 1. p(3ʹ UTR 10) = 0.0271(*), p(3ʹ UTR 50) = 0.013 (*), p(3ʹ UTR 100) = 0.0068 (**), p(3ʹ UTR 250) = 0.0009 (***), and p(3ʹ UTR 500) = 0.0071 (**). e RT-qPCR measurement of sfGFP transcripts in ZSWIM8 KO and Scr cells containing the sfGFP reporters. sfGFP transcript levels are normalized to GAPDH mRNA and the value in Scr cells expressing CDS trigger is set to 1. f Northern blot analyses of miR-16 in sfGFP reporter-expressing cells with ZSWIM8 KO. miR-7 is used as the positive control for ZSWIM8 KO, and miR-92a is used as a loading control. miRNA abundance is normalized to miR-92a. The miRNA abundance in Scr cells containing the sfGFP CDS reporters is set to 1. g The quantification of northern blots in (f). miR-16 levels are normalized to miR-92a and the value in Scr cells without Dox treatment is set to 1. p(lane 7/lane1)=0.0009 (***), p(lane 10/lane4)=0.0007 (***), p(lane8/lane7)=0.0384(*), p(lane9/lane7)=0.0099(**), p(lane11/lane10)=0.0036 (**), and p(lane12/lane10)=0.0042(**). h RT-qPCR measurement of the levels of AGO-associated sfGFP transcripts. RNA was extracted from AGO-IP in cells expressing the CDS or 3ʹ UTR trigger, with or without Dox induction. Statistically significant changes compared with the GFP control are indicated, p = 0.0079 (**). i RT-qPCR measurement of the levels of sfGFP transcripts. RNA was extracted from cells expressing the CDS or 3ʹ UTR trigger, with or without Dox induction. p = 0.0061(**). sfGFP transcript levels are normalized to GAPDH and the value in uninduced cells expressing the CDS trigger is set to 1. (Fig. 2e, h, i). All data are presented as mean ± SD (n = 3 biological replicates). Unpaired two-tailed t test calculated the p values, ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Source data are provided as a Source Data file.