Figure 3.

Gis2p and ZNF9 bind specifically to GWW repeats in RNA and to G-rich sequences in ssDNA. RNA-protein complexes formed between biotinylated RNAs and yeast extracts expressing Gis2-TAP or ZNF9-TAP (eGis2/eZNF9) or recombinant Gis2-His or ZNF9-His purified from E. coli (pGis2/pZNF9) were captured with streptavidin beads and visualized by immunoblot analysis with specific antibodies detecting the TAP or His tag. Representative experiments from at least three biological replicates are shown. (a) RNA pull-downs with short biotinylated RNAs bearing different nucleotide triplet repeats (lanes 2 to 7). The consensus sequence for protein-RNA interaction is depicted on the right. (b) Testing different sizes of GWW loops for interaction with Gis2p/ZNF9 (lanes 1 to 6). The predicted stem-loop structure with varying sizes of GWW-loops is shown to the left. (c) RNA pull-downs after the addition of ten-fold excess of non-labeled competitor RNA (lanes 3 to 5) or ssDNA (lanes 6, 7, and 12 to 14). No RNA was added to control for unspecific binding of proteins to the beads (lanes 8 and 11). (d) Binding ZNF9 to human RNAs containing at least three GWW repeats in the coding region (lanes 2 to 4). (GAUGAA)₅ was used as positive control (lane 5), and (GAUGCU)₅ as negative control (lane 7). Binding of ZNF9 to MYH4 RNA was efficiently competed with ten-fold excess of unlabeled (GAUGAA)₅ RNA (lane 6). A reaction without RNA is shown in lane 8.