Fig 2: Translational readthrough in AGO1 (related to Fig 1) Comparison of efficiency of translational readthrough of AGO1 with that of VEGFA. The assay was done as described in Fig 1C. **P = 0.0051 (with Welch's correction); ***P = 0.002. Numbers indicate % of readthrough.Significance of the identity of the canonical stop codon in AGO1 translational readthrough. This in vitro assay was done as described in Fig 1E. ***P < 0.0001.Demonstration of translational readthrough of AGO1 using Myc-tag reporter assay. Plasmids containing in-frame AGO1-TGA (or GCA)-ISR-Myc/His were transfected in HEK293 cells. His-tagged proteins were enriched using the HisPur Ni-NTA resin, and the readthrough product was detected by Western blot.Data information: Bar graphs (mean ± SE) are representative of at least two independent experiments. Statistical significance was calculated using Student's t-test. ISR, inter-stop codon region; ?ISR, construct without ISR.

Fig 3: Exogenous Ago1x can load miRNAs onto target mRNA in AGO1 knockdown cells (related to Fig 5) Western blot showing the knockdown of AGO1 in HeLa cells. Cells were stably transfected with shRNA that targets the distal 3'UTR of AGO1.qRT–PCR results showing the enrichment of three miRNAs in FLAG-HA-Ago1x immunoprecipitate relative to that in FLAG-HA-Ago1 immunoprecipitate in AGO1 knockdown HeLa cells. Bars: mean ± SE (N = 3)RT–PCR results showing MYC mRNA co-immunoprecipitated with FLAG-HA-Ago1 or FLAG-HA-Ago1x in AGO1 knockdown HeLa cells.Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with FLAG-HA-Ago1 and FLAG-HA-Ago1x in AGO1 knockdown HeLa cells. Source data are available online for this figure.

Fig 4: Ago1x can load miRNAs onto target mRNA Linear regression analysis of miRNAs co‐immunoprecipitated with Ago1 and those co‐immunoprecipitated with Ago1x in HeLa cells. Reads‐per‐million (RPM) values of 395 miRNAs that were common to Ago1 and Ago1x are shown.qRT–PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show the mean of three experiments ± SE.RT–PCR results showing PTEN and MYC mRNAs co‐immunoprecipitated with Ago1 or Ago1x in HeLa cells.Result of co‐immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells. Source data are available online for this figure.

Fig 5: Schematic showing Ago1x‐mediated negative feedback regulation of miRNA pathwayTranslation termination at the first stop codon of AGO1 generates canonical Ago1 isoform. Ago1 can load miRNAs on target mRNAs and repress their expression. Translation readthrough at the first stop codon and termination at the downstream in‐frame stop codon generates Ago1x. let‐7a miRNA promotes this process. Ago1x can load miRNAs on target mRNAs, but it cannot bring down their expression because of its inability to recruit GW182. Thus, it can act as a competitive inhibitor and de‐repress gene expression.