Pol II accumulates near the 5′ end of genes.

<p>(A, C, E, G) The locations of primers used in this assay are shown under the gene structures. (B, D, F, H) HeLa cells were treated with 30 ng/ml of SSA for 2 hours, and the distributions of Pol II were analyzed by chromatin immunoprecipitation (ChIP). The amount of immunoprecipitated DNA fragments was measured by quantitative PCR (upper panels). The ratios between the percent input of SSA-treated cells (SSA) and methanol-treated cells (MeOH) were calculated (MeOH  = 1) (lower panels). Error bars indicate s.d. (n = 4). Statistical significance was investigated by the t-test (*: p<0.05; **: p<0.01).</p

SSA treatment causes splicing inhibition and 3′-end down-regulation.

<p>(A, B, C) HeLa cells were treated with the indicated concentrations of SSA for 4 hours, and RNAs were labeled during transcription with EU between 3 and 4 hours after the addition of SSA (A). Labeled RNAs were analyzed by quantitative RT-PCR to measure the amounts of spliced and unspliced mRNA. Relative splicing activity was defined as the ratio of spliced to unspliced mRNA (methanol-treated cells [Ctrl]  = 100%) (B). The levels of the 5′ and 3′ ends of <i>CDK6</i> (Exon 2 and Exon 8, respectively) were measured by quantitative RT-PCR (C, upper panel), and the ratio between the 3′ and 5′ levels was calculated (methanol-treated cells [Ctrl]  = 100%) (C, lower panel). (D, E, F) HeLa cells were treated with 100 ng/ml of SSA, and RNAs were labeled during transcription with EU for 1 hour as indicated (D). Labeled RNAs were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098015#pone-0098015-g002" target="_blank">Figure 2B and 2C</a> (methanol-treated cells [Ctrl]  = 100%) (E, F). (G, H, I) HeLa cells were treated with 100 ng/ml of SSA for 2 hours, and then washed with fresh medium. Cells were cultivated in fresh medium, and RNAs were labeled during transcription with EU for 1 hour as indicated (G). Labeled RNAs were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098015#pone-0098015-g002" target="_blank">Figure 2B and 2C</a> (methanol-treated cells [Ctrl]  = 100%) (H, I). Error bars indicate s.d. (n = 3). Statistical significance was investigated by the t-test (*: p<0.05; **: p<0.01; ***: p<0.001).</p

3′-end down-regulation by SSA treatment is observed in exosome deficient cells.

<p>(A, B) HeLa cells were transfected with Control or <i>RRP4</i> siRNA (20 nM), and then cultured for 48 hours after transfection. Cells were then treated with the indicated concentration of SSA for 4 hours, and RNAs were labeled during transcription with EU between 3 and 4 hours after the addition of SSA. (A) Half of the transfected cells were analyzed by Western blotting to measure the level of RRP4 protein. Molecular weights are indicated to the left of the gels. (B) RNA samples were purified from the other half of the cells, and labeled RNAs were analyzed by quantitative RT-PCR. The ratios between the 3′ and 5′ ends of <i>CDK6</i>, <i>SMEK2</i>, <i>VEGFA</i> and <i>EGFR</i> were calculated (methanol-treated and control siRNA-transfected cells  = 100%). Error bars indicate s.d. (n = 3). Statistical significance (RRP4 siRNA, 0 ng/ml SSA vs. RRP4 siRNA, 30 ng/ml SSA) was investigated by the t-test (**: p<0.01; ***: p<0.001).</p

PCPA does not cause 3′-end down-regulation in SSA- and U2 AMO–treated cells.

<p>(A, B) HeLa cells were transfected with varying concentrations of U2 AMO (A) or U1 AMO (B) as indicated, and then cultured for 6 hours after transfection. RNAs were labeled during transcription with EU between 5 and 6 hours after transfection, and labeled RNAs were analyzed by quantitative RT-PCR. The levels of the 5′ ends of CDK6 gene (CDK6 Exon 2) and the 3′ ends of CDK6 gene (CDK6 Exon 8) were measured (upper panels), and the ratio between the 3′ and 5′ levels was calculated (methanol-treated cells [Ctrl]  = 100%) (lower panels). Error bars indicate s.d. (n = 3). Statistical significance was investigated by the t-test (*: p<0.05; **: p<0.01; ***: p<0.001). (C) HeLa cells were treated with MeOH (vehicle) or SSA (100 ng/ml), or transfected with Control (3 nmol), U1 (3 nmol), or U2 AMO (10 nmol), and then cultivated for 6 hours. RNAs were labeled during transcription with EU between 5 and 6 hours after the initiation of treatment, and labeled RNAs were analyzed by 3′-RACE. Band sizes (bp) are indicated to the left of the gels. Bands representing RNA that has undergone PCPA (Arrowheads) and spliced mRNA (*) are indicated to the right of the gels.</p

U2 snRNP Is Required for Expression of the 3′ End of Genes

<div><p>Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3′-end down-regulation. Removal of SSA from the culture media restored expression of the 3′ ends of genes, suggesting that U2 snRNP is required for expression of the 3′ end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5′ end of 3′-end down regulated genes, such as <i>CDK6</i>, <i>SMEK2</i> and <i>EGFR</i>, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.</p></div