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

abstract: ...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 CDK6 , SMEK2 and EGFR , 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.

..., 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 CDK6 , SMEK2 and EGFR , indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full

... in the transcriptome of SSA-treated cells, we analyzed RNAs from three independent experiments using Affymetrix human exon arrays. Out of 230,000 probe sets on the array, more than 170,000 probe sets with small standard deviations, corresponding to 15,800 genes, were included in the analysis. To provide illustrative examples, the expression level of each probe set of the CDK6 , SMEK2 and C-MYC genes in SSA-treated cells, relative to the corresponding level in control cells, is shown in Figure 1AC . The expression levels of exons of CDK6 and SMEK2 gradually decreased from the 5 end to the 3 end, whereas the exons of C-MYC did not exhibit this property ( Fig. 1AC ). This phenomenon was also confirmed by RT-qPCR ( Fig. 1DF ). SSA treatment has been reported to cause alternative splicing [19] ; however, we observed no exon skipping in SSA-treated cells, at least in these three genes. 10.1371/journal.pone.0098015.g001 Figure 1 Selected genes exhibiting 3-end down-regulation in SSA-treated cells

... expression level of the 5 end ( CDK6 Ex2) declined gradually as the concentration of SSA increased; however, the level of the 3 end ( CDK6 Ex8) decreased more sharply than that of the 5 end ( Fig. 2C ). Consequently, the ratio between the 3 and 5 levels was decreased by SSA treatment in a dose-dependent manner ( Fig. 2C ). The pattern of splicing activity was very similar to that of the relative level of the 3 end ( Fig. 2B and 2C ). In addition to CDK6 , SMEK2 and another 3-end down-regulated gene

... and S 4). These results suggest that inhibition of U2 snRNP and splicing by SSA is the cause of gene-specific 3-end down-regulation. Therefore, we next investigated in detail the relationship between U2 snRNP activity and 3-end down-regulation. Because CDK6 and SMEK2 exhibited 3-end down-regulation but C-MYC did not, we speculated that CDK6 and SMEK2 have introns that are sensitive to U2 snRNP inhibition by SSA, and the splicing level after SSA treatment might differ among these three genes. Alternatively, splicing level might decrease from the 5 to 3 ends of genes, and this could contribute to 3-end down-regulation. To test these possibilities, we measured the relative splicing levels of CDK6 , SMEK2 and C-MYC , and observed significant splicing inhibition in all introns we tested. The strongest splicing inhibition was observed on C-MYC , which is not 3-end down-regulated ( Fig. S5 ). We also found that splicing level did not decrease from the 5 to the 3 end ( Fig. S5 ). These results suggest

... targeting RRP4 mRNA, which encodes a component of the exosome complex [21] . After treatment with siRNAs against RRP4 , the RRP4 protein level significantly decreased ( Fig. 4A ). We observed a significant 3-end down-regulation of SMEK2 and EGFR , another 3-end down-regulated gene, upon SSA treatment of RRP4-knockdown cells ( Fig. 4B and S 1B), suggesting that 3-end down-regulation occurs even in the exosome deficient cells. CDK6 and VEGFA also showed 3-end down-regulation by SSA treatment in RRP4

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Gene: SMEK2 - SMEK homolog 2, suppressor of mek1 (Dictyostelium) - (NCBI)

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