FIGURE 8.

The UTP effect involves specific action of the TbMP57 TUTase. (A–C) RNAi to TbMP81/b-II. (A) Assay of the specific TUTase step in U-insertion, using precleaved U-insertion substrate (Igo et al. 2000) and 0.25 mM PPi, catalyzed by rapid cell lysates of TbMP81/b-II RNAi cells (b-IIi; Law et al. 2005) and control cells (Co; 29.13). The (5′-end-labeled) upstream input oligoribonucleotide (5′ in; 18 nt) and the guided +1 and +2 U-additions are indicated. (B) Assay of nonspecific U-addition activity of the TbMP57 TUTase in the lysates of A (see Law et al. 2005). In this experiment, the unlabeled input RNA is a mixture of two size classes, and the two indicated bands both represent molecules that acquired one U from the added radioactive [α-³²P]UTP (Law et al. 2005), while nonspecific U-addition activity of the TbTUT108 should instead generate a ladder of longer fragments (Aphasizhev et al. 2002, 2003b; Ernst et al. 2003). (C) UTP effect assayed in precleaved U-deletion reactions as in Figure 3B ▶, except using the same lysates as in A and B, with 0.25 mM PPi.