FIGURE 2.

U1 70-kDa silencing destabilizes the level of U1 snRNA and inhibits cis- but not trans-splicing. A, growth curves of T. brucei cells silenced for the U1 70 kDa. Growth of uninduced cells was compared with growth after tetracycline addition. Both uninduced and induced cultures were diluted daily to 2 × 10⁴ cells per ml. B, Northern blot analysis of the U1 70-kDa gene. RNA was prepared from uninduced cells (−Tet) and from cells after 2 days of induction (+Tet). Total RNA (20 μg) was subjected to Northern analysis with randomly labeled probes. The transcript of the U1 70 kDa and the dsRNA are shown, as well as 7SL RNA, which was used to control for equal loading. C, silencing of the U1 70 kDa specifically destabilized the level of U1 snRNA. Total RNA was extracted from uninduced cells or after 2 days of induction. The level of the U snRNAs was determined by primer extension, and the degree of reduction was determined by phosphorimaging. D, effect of U1 70-kDa silencing on cis-splicing. cDNA was prepared from the RNA extracted from uninduced cells (−Tet) or after 2 days of induction (+Tet). The cDNA was subjected to PCR with oligonucleotides 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, reduction in the level of U1 70 kDa during silencing of gene expression. Proteins from cells (10⁷) after 2–5 days of silencing were separated on a 10% SDS-polyacrylamide gel and subjected to Western analysis with anti-U1 70-kDa antibodies prepared as described under “Experimental Procedures.” An additional aliquot of tetracycline was added after 3 days to induce maximum silencing. Reactivity with PTB1 antibodies (59) was used as a control for equal loading. Panel b, trans-splicing during U1 70-kDa silencing. 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 snoRNA was used to control for the level of the RNA in the samples. The products were separated on a 6% acrylamide denaturing gel. The results were subjected to phosphorimaging, and quantitation was performed 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 the U1 70 kDa. 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.