CCNE1 and E2F1 Partially Suppress G1 Phase Arrest Caused by Spliceostatin A Treatment

The potent splicing inhibitor spliceostatin A (SSA) inhibits cell cycle progression at the G1 and G2/M phases. We previously reported that upregulation of the p27 cyclin-dependent kinase inhibitor encoded by CDKN1B and its C-terminal truncated form, namely p27*, which is translated from CDKN1B pre-mRNA, is one of the causes of G1 phase arrest caused by SSA treatment. However, the detailed molecular mechanism underlying G1 phase arrest caused by SSA treatment remains to be elucidated. In this study, we found that SSA treatment caused the downregulation of cell cycle regulators, including CCNE1, CCNE2, and E2F1, at both the mRNA and protein levels. We also found that transcription elongation of the genes was deficient in SSA-treated cells. The overexpression of CCNE1 and E2F1 in combination with CDKN1B knockout partially suppressed G1 phase arrest caused by SSA treatment. These results suggest that the downregulation of CCNE1 and E2F1 contribute to the G1 phase arrest induced by SSA treatment, although they do not exclude the involvement of other factors in SSA-induced G1 phase arrest.

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns

Chemical splicing modulators that bind to the spliceosome have provided an attractive venue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. Yet, the biological effect of proteins containing translated intron sequences remains unclear. Here we identified a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) using genome-wide ribosome profiling and bio-orthogonal non-canonical amino-acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through JNK phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-Based proteomic analysis of mRNA splicing relevant proteins in aging HSPCs

Background: HSPC aging was closely associated with the organism aging, senile diseases and hematopoietic related diseases. Therefore, study on HSPC aging born great significance to further elucidate the mechanisms of aging and to treat hematopoietic disease resulted from HSPC aging. Little attention had been paid to mRNA splicing as a mechanism underlying HSPC senescence. Results: We used our lab’s patented aging model of HSPCs in vitro to analyze mRNA splicing relevant proteins alterations with iTRAQ based proteomic analysis. We found that not only the notable mRNA splicing genes such as SR, hnRNP, WBP11, Sf3b1, Ptbp1 and U2AF1 but also the scarcely reported mRNA splicing relavant genes such as Rbmxl1, Dhx16, Pcbp2, Pabpc1 were significantly down-regulated. We further verified their genes expressions by qRT-PCR. In addition, we reported the effect of Spliceostatin A (SSA), which inhibits mRNA splicing in vivo and in vitro, on HSPC aging. Conclusions: It was concluded that mRNA splicing emerged as an important vulnerability of HSPC aging. This study improved our understanding of the role of mRNA splicing in the HSPC aging process.