Spliceostatin A

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

Spliceostatin C (SPC) is a component of a bioherbicide isolated from the soil bacterium Burkholderia rinojensis. The chemical structure of SPC closely resembles spliceostatin A (SPA) which was characterized as an anticancer agent and splicing inhibitor. SPC inhibited the growth of Arabidopsis thaliana seedlings with an IC50 value of 2.2 µM. The seedlings exposed to SPC displayed a significant response with decreased root length and number and inhibition of gravitropism. Reverse transcriptase semi-quantitative PCR (RT-sqPCR) analyses of 19 selected genes demonstrated the active impact of SPC on the quality and quantity of transcripts that underwent intron rearrangements as well as up or down expression upon exposure to SPC. Qualitative and quantitative proteomic profiles identified 66 proteins that were significantly affected by SPC treatment. Further proteomics data analysis revealed that spliceostatin C induces hormone-related responses in Arabidopsis seedlings. In silico binding studies showed that SPC binds to a pocket between the SF3B3 and PF5A of the spliceosome.

Pre-mRNA splicing is one of the most important mechanisms in gene expression in eukaryotes, and therefore splicing inhibition affects various cellular functions. We previously reported that the potent splicing inhibitor spliceostatin A (SSA) causes cell cycle arrest at G1 and G2/M phases. Upregulation of the p27 cyclin dependent kinase inhibitor, encoded by the CDKN1B gene, is one of the reasons for G1 phase arrest caused by SSA treatment. However, the molecular mechanism of p27 upregulation by SSA remains unknown. In this study, we found that SSA treatment caused stabilization of the p27 protein and increase of CDKN1B mRNA. SSA did not affect transcription of CDKN1B gene, but stabilized CDKN1B mRNA. Finally, we revealed that the 3' untranslated region of CDKN1B mRNA was involved in the stabilization. These results suggest that stabilization of CDKN1B mRNA is one of the reasons of upregulation of the p27 protein by SSA.