Figure 2

Specific binding of Tip110 protein to U6 RNA. a Complex formation between Tip110 and U6 RNA. ³²P-labeled U6 RNA (*U6) was incubated with 1, 5, 10, and 100 ng recombinant GST (lanes 2–5) or GST-Tip110 (lanes 6–9). The reaction containing no recombinant protein was included to show free ³²P-labeled U6 RNA (lane 1). The RNA–protein complex formation was determined using the electrophoretic mobility shift assay (EMSA). b Specificity of Tip110 binding to U6 RNA. ³²P-labeled U6 RNA (*U6) was incubated with 10 ng recombinant GST-Tip110 in the presence of 5, 10, and 50 ng unlabeled U6 RNA (lanes 3–5), yeast tRNA (lanes 6–8), or polyinosinic:polycytidylic acid (poly I.C) (lanes 9–11). The complex formation of Tip110 and U6 RNA was determined using the EMSA. The binding reactions containing *U6 only (lane 1) and *U6 and GST-Tip110 (lane 2) were also included. c Binding of Tip110 mutants to U6 RNA. ³²P-labeled U6 RNA (*U6) was incubated with 10 ng recombinant GST-Tip110s (lane 3), GST-ΔNLS (lane 4), or GST-ΔRRM (lane 5). The RNA–protein complex formation was determined using the EMSA. The binding reactions containing *U6 RNA only (lane 1) or *U6 RNA and GST-Tip110 (lane 2) were also included. C RNA–protein complex; F unbound free RNA.