FIG 3.

RNA binding and annealing activity of TbRGG3. (A) UV cross-linking assays were performed using 5 fmol of radiolabeled guide RNA (gA6[14]) or a fragment of preedited A6 mRNA (A6U5) with either 500 nM or 1 μM purified GST-TbRGG3. Reaction mixtures were subjected to UV cross-linking and treated with RNase A, and proteins were resolved by 10% SDS-PAGE. Proteins that cross-linked to radiolabeled RNA were visualized by PhosphorImager analysis. TbRGG2 binding was performed in parallel as a positive control, while reactions using mixtures containing no protein (−) were performed as a negative control. (B) Competition experiments were performed by incubating 7.5 pmol of unlabeled homoribopolymer RNA with GST-TbRGG3 prior to the addition of radiolabeled gRNA (gCYb[558]). Reaction mixtures were UV cross-linked and visualized as stated above. (C) RNA annealing assays were performed with 10 nM radiolabeled A6U5 41-nt pre-mRNA and 10 nM unlabeled gA6[14] gRNA at the indicated protein concentrations. Negative-control reactions were performed in the absence of gRNA (−) or with both RNAs in the absence of protein (+). The reaction mixture containing p22 served as a negative control. Reaction mixtures were incubated for 20 min, treated with proteinase K, and analyzed by 8% native PAGE.