Supplementary Figure 4: Functional test for Ago2 or GW182 dependency and for CDS-targeted miRNA to induce translation abortion.
From: A novel class of microRNA-recognition elements that function only within open reading frames
a, One miR-20a MRE and four Let-7b MREs were tested in wild-type or Ago2 knockout MEFs. Note that Ago2 knockout MEFs contain a small population of undeleted cells, which likely contributed to the basal level of Ago2, as shown by western blotting on top. The MREs inserted in both CDS (black bar) or 3’UTR (grey bar) were tested. b, One miR-20a MRE and four Let-7b MREs were tested in non-specific siRNA-treated (NC) or siGW182-treated HeLa cells. Knockdown efficiency was determined by western blotting, as shown on top. The MREs inserted in both CDS (black bar) or 3’UTR (gray bar) were tested. The data shown in a and b were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). c, Western blotting analysis of endogenous proteins in non-specific siRNA-treated (-) or siGW182-treated (+) HeLa in response to miR-20a mimic (left) or antagomir (right). d, Repeated experiments as in main Fig. 4b. Western blotting analysis of Ago2 and GW182 associated with the Renilla luciferase reporter carrying perfect seed base-pairing (“seed”) or imperfect seed with or without additional 3nt mismatches in 3’ end (“3p3MM+seed” and “3p3MM”) in CDS or 3’UTR. Equal amounts of captured reporter mRNAs were detected by RT-PCR and the Firfly reporter served as a negative control. e, Repeated experiments as in main Fig. 5b. Western blotting of the FLAG-tagged luciferase reporter proteins expressed in transfected HeLa cells. The second to last lane was from cells co-transfected with the reporter containing the miR-20a MRE and the miR-20a antagomir. f, Repeated experiments as in main Fig. 5c. Western blotting of GFP-2A-MRE-2A-mCherry reporter proteins expressed in transfected HeLa cells. Co-transfected FLAG-tagged Renilla served as a loading control.
