FIGURE 6.

Trans-silencing capacity of tsRNAs (dual luciferase assay). (A) Cand14-mediated trans-silencing. Dual luciferase assay with the Renilla luciferase reporter gene carrying a fully cand14-complementary target site in its 3′ UTR (psi-cand14). Addition of a cand14 antisense molecule increased Renilla luciferase expression, as expected, if cand14 had RNAi-like trans-silencing capacity. Likewise, overexpression of Ago2 enhanced cand14-mediated trans-silencing. The specificity of the de-repression with anti-cand14 was confirmed with three (antisense) control oligonucleotides (anti-con1, anti-con2, anti-con3). (B) Cand45 overexpression from a plasmid into which the genomic sequence of cand45 had been cloned (cand45-45; by Northern blot). Cand45-empty: cand45 cloning plasmid with only a cloning site between the cand45 RNaseZ cleavage site and the RNA PolIII terminator; cand45-con: cand45-derived expression plasmid in which the cand45 sequence in cand45-45 was replaced with an arbitrary control sequence; M: Decade (Ambion) RNA size marker. (C) Cand45 overexpression (cand45-45)-dependent, anti-cand45 oligo-induced trans-silencing in HCT116 cells. Dual luciferase assay with reporter gene carrying a fully cand45-complementary target site in the Renilla luciferase 3′ UTR (psi-cand45). Cand45-empty, cand45-con, cand45-45 as in C. (D) Confirmation of the specificity of the anti-cand45 induced trans-silencing effect in panel D by the use of three additional (antisense) control oligonucleotides (anti-con1, anti-con2, anti-con3). (E) Anti-cand45 induced trans-silencing is RNAi-related. Cand45 overexpression (cand45-45)-dependent, anti-cand45 (“anti-45”)-induced trans-silencing is enhanced by overexpression of slicing-competent Argonaute 2, but mitigated by overexpression of the nonslicing Argonautes 1, 3, and 4. (F) Predicted cand45:anti-cand45 duplex; A.U.: arbitrary units; error bars indicate standard deviation from n = 3 transfections.