FIG. 5.

TbMP18 is essential for both U-deletional and U-insertional cleavages. (A and B) Assessments of specific editing cleavage activities at U-deletional (A) and U-insertional (B) editing sites. (The fainter 34-nt product above the main band in panel B represents cleavage at the next U-insertional editing site.) The reactions contain and lack AMP-CP, respectively. (C and D) Assessments of the basic activities of the U-deletional (C) and U-insertional (D) endonucleases. The assay for the latter generates two main products (26). These experiments used rapid extracts prepared from control or RNAi cells induced for 3 or 6 days, as indicated. In this and subsequent experiments, the protein amount analyzed is indicated below the extract designation and 2.4 μg was used as 1× (unless otherwise noted). G and nt, G and nucleotide ladders from treatment of this mRNA with NaOH and RNase T1; −, no extract; arrowhead, the expected product (size shown below). In the substrate diagrams of this figure and subsequent figures, the asterisk represents the ³²P label of the mRNA strand.