Daisuke Kaida

Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA

The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing†‡

ABSTRACT Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.

Yeast Whi2 and Psr1‐phosphatase form a complex and regulate STRE‐mediated gene expression

Background:  In response to various stressful situations, including diauxic conditions, the Msn2 and Msn4 transcription factors induce STRE‐mediated gene expression of many stress responsive genes in Saccharomyces cerevisiae. This is called the general stress response. The whi2 cells in the stationary phase are smaller than wild‐type cells.

The PY-motif of Bul1 protein is essential for growth of Saccharomyces cerevisiae under various stress conditions.

The previously identified BUL1 gene was found to encode a protein bound to Rsp5-ubiquitin ligase in budding yeast. We have identified the BUL2 gene as a functional homologue of BUL1. The bul1 bul2 double disruptant was sensitive to various stresses, such as high temperature, salts, and a non-fermentable carbon source. Each Bul protein has a putative PY-motif that has been predicted to interact with one of three WW-domains of Rsp5. A mutant Bul1 containing an altered PY-motif was defective in ability to bind to Rsp5 in the two-hybrid system and hardly co-immunoprecipitated with Rsp5. Furthermore, the mutant was not able to overcome all growth defects of the double disruptant. Thus, Bul proteins are essential for growth in various stress conditions, and their functions are mediated through the PY-motif, probably by binding to Rsp5.

Splicing in oncogenesis and tumor suppression

Post‐transcriptional modifications, such as 5′ end capping, 3′ end polyadenylation and splicing, are necessary for the precise regulation of gene expression and transcriptome integrity. Therefore, it is not surprising that abnormalities of these post‐transcriptional modifications prompt numerous diseases, including cancer. In fact, many studies revealed that misregulation of mRNA processing, especially splicing, are observed in a variety of cancer cells. In this review we describe how changes within RNA splicing regulatory elements or mutations in the processing factors alter the expression of tumor suppressors or oncogenes with pathological consequences. In addition, we show how several small molecules that bind to spliceosomal components and splicing regulators inhibit or modulate splicing activity. These compounds have anticancer activity and further development of small molecule modulators has potential in next generation cancer therapy.

Splicing inhibition decreases phosphorylation level of Ser2 in Pol II CTD

Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II (Pol II), especially Ser2 and Ser5 residues, plays important roles in transcription and mRNA processing, including 5′ end capping, splicing and 3′ end processing. These phosphorylation events stimulate mRNA processing, however, it is not clear whether splicing activity affects the phosphorylation status of Pol II. In this study, we found that splicing inhibition by potent splicing inhibitors spliceostatin A (SSA) and pladienolide B or by antisense oligos against snRNAs decreased phospho-Ser2 level, but had little or no effects on phospho-Ser5 level. In contrast, transcription and translation inhibitors did not decrease phospho-Ser2 level, therefore inhibition of not all the gene expression processes cause the decrease of phospho-Ser2. SSA treatment caused early dissociation of Pol II and decrease in phospho-Ser2 level of chromatin-bound Pol II, suggesting that splicing inhibition causes downregulation of phospho-Ser2 through at least these two mechanisms.

Rsp5–Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

The reciprocal regulation between splicing and 3'-end processing.

Most eukaryotic precursor mRNAs are subjected to RNA processing events, including 5′‐end capping, splicing and 3′‐end processing. These processing events were historically studied independently; however, since the early 1990s tremendous efforts by many research groups have revealed that these processing factors interact with each other to control each others functions. U1 snRNP and its components negatively regulate polyadenylation of precursor mRNAs. Importantly, this function is necessary for protecting the integrity of the transcriptome and for regulating gene length and the direction of transcription. In addition, physical and functional interactions occur between splicing factors and 3′‐end processing factors across the last exon. These interactions activate or inhibit splicing and 3′‐end processing depending on the context. Therefore, splicing and 3′‐end processing are reciprocally regulated in many ways through the complex protein–protein interaction network. Although interesting questions remain, future studies will illuminate the molecular mechanisms underlying the reciprocal regulation. WIREs RNA 2016, 7:499–511. doi: 10.1002/wrna.1348

U2 snRNP is required for expression of the 3' end of genes.

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.

Upregulation of p27 cyclin-dependent kinase inhibitor and a C-terminus truncated form of p27 contributes to G1 phase arrest

Potent anti-cancer compounds FR901464 and its methyl-ketal derivative spliceostatin A (SSA) inhibit cell cycle progression at G1 and G2/M phases. These compounds bind to the spliceosome and inhibit the splicing reaction. However, the molecular mechanism underlying G1 arrest after SSA treatment remains unknown. In this study, we found that ~90% of SSA-treated cells arrested at G1 phase after cell cycle synchronization. SSA treatment caused upregulation of the p27 cyclin-dependent kinase inhibitor both at mRNA and protein levels. In addition to p27, we observed expression of p27*, a C-terminal truncated form of p27 that is translated from CDKN1B (p27) pre-mRNA accumulated after splicing inhibition. Overexpression of p27 or p27* inhibited the exit from G1 phase after a double thymidine block. Conversely, knocking down of p27 by siRNA partially suppressed the G1 phase arrest caused by SSA treatment. There results suggest that G1 arrest in SSA-treated cells is caused, at least in part, by upregulation of p27 and p27*.