Fig. 1: An evolutionarily conserved role of GW182 in circular RNA degradation.

a To measure RNA half-lives, Drosophila DL1 cells were treated with actinomycin D for the indicated amounts of time. qRT-PCR was then used to quantify expression of endogenous circRNA and its parental linear RNA. Data were normalized to the RNA levels observed at 0 h actinomycin D treatment sample. n = 3. b qRT-PCR quantification of endogenous circdati and circlaccase2 in RNA purified from DL1 cells that were treated with the indicated dsRNAs for 3 days. c DL1 cells were treated with β-gal dsRNA (as a control) or GW182 dsRNA for 3 days. qRT-PCR was then performed to measure expression of the indicated steady-state circRNAs. d After dsRNA treatment, 250 µM 4sU was added to the cells to label nascent transcripts for 5 min prior to RNA isolation. qRT-PCR was then employed to measure levels of nascent circRNAs. Data throughout (b–d) were normalized to the β-gal dsRNA sample. n = 3. e The abundance of high confidence circRNAs (TPM ≥ 0.1) in Drosophila S2 cells was measured by RNA-seq upon GW182 depletion. Each dot represents one circRNA. f Boxplot shows that levels of the high confidence circRNAs significantly increased in GW182-depleted cells while their parental mRNAs abundance decreased slightly. g Cumulative distribution functions of circRNA junction read ratio for each backsplicing site in β-gal dsRNA or GW182 dsRNA sample. Statistical significances of data throughout (f, g) were computed using the Mann–Whitney U test. n = 3. h Drosophila S2 cells were treated with β-gal dsRNA or two independent GW182 dsRNAs for 3 days. qRT-PCR quantification was then used to measure levels of endogenous circRNAs in RNA purified from nuclear (Nuc) or cytoplasmic (Cyto) fractions. n = 4. i The similarity (similar amino acid properties) between Drosophila GW182 and its human homologs. j Human HeLa cells were treated for 48 h with a control siRNA or specific siRNAs to knockdown TNRC6A, TNRC6B or TNRC6C. qRT-PCR was then performed to measure levels of the indicated human circRNAs. Data were normalized to the control siRNA sample. n = 3. k Schematic representation of Drosophila GW182. Expression plasmids of GW182 mutants were generated. l–o Overexpression of GW182 mutants to identify the key domain of GW182 in circRNA degradation. l and n western blotting was used to examine expression of the Flag-tagged GW182 proteins. α-Tubulin was used as a loading control. Representative blots are shown. n = 3. m and o qRT-PCR quantification of Drosophila endogenous circRNAs in RNA purified from S2 cells that were transfected with wild type (WT) or the indicated mutants of GW182 plasmids for 48 h. m n = 3. o n = 4. Data were normalized to the empty vector (EV) sample. All data are shown as mean ± SD. ^∗∗P < 0.01; ^∗P < 0.05