Fig 1: Upstream cleavage products are not bound by PABPC1 or eIF4E and are degraded by both XRNs and the exosome complex. (A and B) Levels of the target RNA upstream cleavage products associated with (A) PABPC1 or (B) eIF4E from untreated cells (gray bars), cells treated with target ASOs or siRNA alone (red bars), or cells treated first with XRN siRNAs and EXOSC5 shRNA and then with ASO or siRNA (blue bars). Levels of the target RNA cleavage products were determined 48 h post treatment by RT-qPCR and are shown as a percentage of their respective untreated controls (transfection reagent only). (C) Cells were treated with EXOSC5 shRNA or XRN siRNAs and EXOSC5 shRNA and then with ASO or siRNA. 3′-RACE of the target RNA upstream cleavage products was performed 48 h post treatment. The PPS used for the RT-qPCR are listed in Supplementary Figure S1A. The adapter and primers used for the 3′-RACE are listed in Supplementary Figure S1C.
Fig 2: Exosome complex degrades upstream cleavage products. (A) Cellular levels of indicated mRNAs 48 h post siRNA treatment. The mRNA levels were determined by RT-qPCR and are shown as a percentage of untreated controls. The mean and standard errors reported are based on three trials. (B) 3′-RACE of the target RNA upstream cleavage products from ASO or siRNA treated and control cells (UTC) treated with transfection reagent only. 3′-RACE was performed 48 h post treatment with Malat 1, α-Actinin or PTEN ASOs or siRNA alone or with both target-specific ASO/siRNA and exosome/exonuclease siRNAs. The predicted sizes of the 3′-RACE products are: Malat1 378; α-Actinin 423; PTEN ASO 439; PTEN siRNA 386. (C) Cellular level of EXOSC5 mRNA 48 h post shRNA treatment. (D) Western blot analysis of EXOSC5 protein from untreated (UTC) and siRNA treated cells 48 h to 144 h post siRNA treatment. (E) 3′-RACE of upstream cleavage products was performed on extracts of cells treated with Malat1, α-Actinin, or PTEN ASOs or siRNA alone or first treated 144 h with EXOSC5 shRNA and then with target-specific ASO/siRNA. The PPS used for the RT-qPCR are listed in Supplementary Figure S1A. The adapter and primers used for the 3′-RACE are listed in Supplementary Figure S1C.
Fig 3: The 3'-5' RNA exosome-mediated, but not the 5'-3' XRN1-mediated, RNA degradation is required for the anti-JEV activity of ZAP.A549 cells with shRNA targeting LacZ, XRN1, and EXOSC5 were transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5. After 10 h of JEV infection (MOI = 5), cells lysates were analyzed by western blot for the indicated proteins (A and D, upper panel). The relative quantification of NS3 normalized by actin was quantified by ImageJ software (A and D, lower panel). Data are mean ± SD (from four independent experiments). Total RNA and culture supernatants were harvested for the measurement of viral RNA by RT-PCR (B and E) and viral titer by plaque assay (C and F). Data are representative results from repeated experiments and shown as mean ± SD (n = 3). The statistical significance was estimated by two-tailed Student’s t test. * P=0.05; ** P=0.01; *** P=0.001; NS, not significant.
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