Preparations of endogenous human exosomes exhibit specific 3′-to-5′ distributive, exoribonucleolytic activity. (A) Structure comparison of 3′-end modified oligos utilized for RNA degradation assays. Substrate 1 is 2′-O-methylated, while substrate 2 is also 3′-phosphorylated. Both substrates are labeled with 6-FAM (6-carboxyfluorescein) at the 5′-end. (B) RNA degradation intermediates resolved by denaturing urea-polyacrylamide gel electrophoresis; ExoI was incubated with either substrate 1 or 2 for the indicated time. (C) RNA degradation intermediates produced by ExoII, incubated with RNA substrates as in B. (D) Panel of assays testing the effect of DTSSP on DIS3 enzymatic activity. (Left) Coomassie stained SDS-PAGE displaying DIS3-3xFlag purification from HEK293 cells (H.Ch. and L.Ch.: IgG heavy and light chains, respectively). (Center) DIS3 incubated with both RNA substrates, as in B. (Right) DTSSP-treated DIS3 was incubated with substrate 1 and the RNA degradation products were separated as in B.
