Abstract
The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.
Highlights
Most protein-coding genes are transcribed into a precursor RNA that requires significant processing to produce mature mRNA
It was recently reported that IGG-induces Activating transcription factor 3 (ATF3) indirectly through ATF4, it was unclear if ATF3 contributed to cell death [13]
ATF3 protein increased in response to both drugs, we detected a greater increase in response to pladienolide B (PB) (Fig 1B)
Summary
Most protein-coding genes are transcribed into a precursor RNA (pre-mRNA) that requires significant processing to produce mature mRNA. One of the more complex modifications is the removal of introns and ligation of exons through the process of pre-mRNA splicing [1]. The spliceosome is composed of five small nuclear RNAs (snRNAs), U1, U2, U4, U5 and U6, which are each associated with specific proteins yielding their corresponding small nuclear ribonucleoprotein complexes (snRNP). These snRNPs bind sequentially to cis-acting elements in pre-mRNA including the 5’ and 3’ splice sites, the branch sequence, and polypyrimidine tract, leading to the assembly of a functional spliceosome through the E, A, B, B and C pre-spliceosome complexes [2].
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