FIGURE 4.

RNAi silencing of U1A suggests its function in both trans- and cis-splicing. A, growth curves of T. brucei cells silenced for U1A. Growth of uninduced cells was compared with growth after tetracycline addition. Both uninduced and induced cultures were diluted daily to 5 × 10⁴ cells per ml. B, Northern blot analysis. 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 the U1A and the dsRNA as well as 7SL RNA that was used to control for equal loading are indicated. C, silencing of U1A has no effect on the level of U1 snRNA. Total RNA was extracted from uninduced cells or after 2 days of induction, and 10 μg was used to determine the level of the U1 snRNA by primer extension. The level of U3 was used to control for equal loading. D, cis-splicing of PAP in U1A silenced cells. 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. E, panel a, effect of U1 silencing on the level of U1A protein. Cells expressing PTP-U1A and the U1A silencing construct were silenced for the number of days indicated. Proteins from cells (10⁷) after 2–5 days of silencing were separated on a 10% SDS-polyacrylamide gel and subjected to Western analysis as described under “Experimental Procedures.” Reactivity with PTB1 antibodies was used as a control for equal loading. Panel b, U1A silencing affects trans-splicing. Total RNA (10 μg) from the same cells as described in panel a was subjected to primer extension with an oligonucleotide complementary to the intron region of SL RNA (supplemental S-1). Primer extension of U3 was used to determine the RNA used. The products were separated on a 6% acrylamide denaturing gel. The results were subjected to phosphorimaging and quantitation using ImageJ. The levels of SL RNA and Y structure are given as fold change with respect to the amount present at day 0 and were normalized to the level of U3 snoRNA. Panel c, changes in the level of tubulin precursor during silencing of U1A. Total RNA (20 μg) from the cells described in panels a and b was subjected to Northern analysis with antisense tubulin RNA probe. The blot was hybridized with random-labeled 7SL RNA probe to control for equal loading. F, localization of U1A with respect to SL RNA. In situ hybridization combined with immunofluorescence was performed as described under “Experimental Procedures.” Panel a, in situ hybridization with SL RNA; panel b, immunofluorescence of PTP-tagged U1A; panel c, merge of panels a and b; panel d, DIC and DAPI-stained nuclei merge with panel c. Panel e, fluorescence emissions at 445–450 nm (blue, DAPI), 525–550 nm (green, the FITC bound to antibody), and 650–690 nm (red, SL RNA, Alexa fluor 647) were examined in the area surrounding the line (inset). The intensity of the different chromophores is plotted, demonstrating overlap between the SL RNA, U1A, and the nucleus signals.