FIGURE 8.

Small RNAs derived from tRNA^Gln poorly associate with Argonaute proteins. (A) small tRNA^Gln RNAs partially cofractionate with Ago2. HeLA S100 extract was fractionated through a Superdex-200 column. Every second fraction was tested for Ago2 and Dicer with Western blotting, for miR-21 and small tRNA^Gln (labeled as tRNA) species with Northern hybridizations. Fractions containing the corresponding size markers (kDa) are indicated with arrows. Vertical bars are representing the level of small tRNA in the corresponding fractions in arbitrary units. (B) RNAs derived from tRNA^Gln incorporate into Ago1 and Ago2 complexes. FLAG immunoprecipitations were carried out from cells that were transfected with empty FLAG, FLAG∷Ago1, and FLAG∷Ago2 fusion plasmids. The total lysate and the bound fractions were assayed with Northern hybridization using complementary RNA to the sequenced small tRNA^Gln fragment as a probe. Black arrows indicate the small RNAs derived from the tRNA. (C) small RNAs derived from tRNA^Gln are resistant to β-elimination. Northern hybridizations show the results of the effect of β-elimination on miR-21 and small RNA processed from tRNA. Black arrows indicate miRNA and small tRNA fragments.