FIGURE 6.

Imp3 is required for U3-18S hybridization. (A) Primer extension analysis of the signal of U3-18S hybridization detected by CMCT modification of the pre-rRNA (ETS-18S). Lane intensity quantification, normalized as in Figure 5A, is shown on the right for lanes containing Imp3 alone (red) or in the presence of either U3 (box A/A′ + hinge) (orange) or U3 (AS box A/A′ + hinge) (cyan). The CMCT reactions were performed as in Figure 5A except that samples were treated with 40 mg/mL CMCT for 1 min at 30°C. Ignore primer extension pausing at C4 (asterisk), which is due to a break in the template. (B) T1 cleavages were detected by monitoring breaks in ³²P-labeled pre-rRNA (IX-18S) in the absence or presence of 0.3 μM Imp3. Lane intensity quantification, normalized to counts in the full-length substrate, is shown on the right for lanes containing pre-rRNA (IX-18S) alone (dashed black) or in the presence of Imp3 incubated with either U3 (box A/A′ + hinge) (orange), U3 (AS box A/A′ + hinge) (cyan), or buffer (red). RNA-protein complexes were first incubated for 45 min, then treated with 1.6 units of T1 in a 20-μL reaction for 15 min at RT. (C) Pre-rRNA (IX-18S) sites whose modification changes upon addition of U3 (box A/A′ + hinge) in the presence of only Imp3 are indicated on the secondary structure of the pre-rRNA: CMCT (open circle) and T1 (bulls eye).