FIGURE 6.

PRP19 is essential for trans-splicing and is localized in the SL RNP factory. A, growth curves of T. brucei cells silenced for PRP19 homologue. Growth of uninduced cells was compared with growth after tetracycline addition. Both uninduced and induced cultures were diluted daily to 5 × 10⁵ cells per ml. B, Northern blot analysis of the prp19 gene. RNA was prepared from uninduced cells (−Tet) and cells after 2 days of induction (+Tet). Total RNA (20 μg) was subjected to Northern analysis with randomly labeled probes. The transcript of PRP19, dsRNA, as well as 7SL RNA that was used to control for equal loading are indicated. C, silencing of PRP19 affects cis-splicing. RNA was prepared from uninduced cells or after 2 days of induction. cDNA was prepared and subjected to PCR amplification with oligonucleotide specific to mature or pre-PAP transcript, as described under “Experimental Procedures.” The level of 7SL RNA was used to control for equal amounts of cDNA. D, silencing of PRP19 affects trans-splicing and SL RNA capping. Panel a, total RNA was extracted from uninduced cells or after 2 days of induction and was subjected to primer extension with oligonucleotide complementary to the intron region of SL RNA (supplemental S-1). The products were separated on a 6% acrylamide denaturing gel. The level of U3 snoRNA was used as a control for equal loading. Panel b, capping of SL RNA is perturbed in PRP19 silenced cells. RNA as in panel a was subjected to primer extension with oligonucleotide complementary to SL RNA (supplemental S-1), and extension products were separated next to the DNA sequence of the SL RNA gene. The positions of the cap nts are indicated. E, localization of PRP19. Transgenic cells for tSNAP42-CFP and YFP-PRP19 were stained with Hoechst. Panels b and c depict an enlargement of the cell located in the left side, and panels d and e show an enlargement of the cell on the right side. The cells were visualized as described under “Experimental Procedures.” Fluorescence image stacks were deconvoluted and rendered as an isosurface model showing overlapping signals tSNAP42 and PRP19. The Hoechst stain is shown in red.