Abstract
Depending on the cell lines and cell types, dimethyl sulfoxide (Me2SO) can induce or block cell differentiation and apoptosis. Although Me2SO treatment alters many levels of gene expression, the molecular processes that are directly affected by Me2SO have not been clearly identified. Here, we report that Me2SO affects splice site selection on model pre-mRNAs incubated in a nuclear extract prepared from HeLa cells. A shift toward the proximal pair of splice sites was observed on pre-mRNAs carrying competing 5'-splice sites or competing 3'-splice sites. Because the activity of recombinant hnRNP A1 protein was similar when added to extracts containing or lacking Me2SO, the activity of endogenous A1 proteins is probably not affected by Me2SO. Notably, in a manner reminiscent of SR proteins, Me2SO activated splicing in a HeLa S100 extract. Moreover, the activity of recombinant SR proteins in splice site selection in vitro was improved by Me2SO. Polar solvents like DMF and formamide similarly modulated splice site selection in vitro but formamide did not activate a HeLa S100 extract. We propose that Me2SO improves ionic interactions between splicing factors that contain RS-domains. The direct impact of Me2SO on alternative splicing may explain, at least in part, the different and sometimes opposite effects of Me2SO on cell differentiation and apoptosis.
Highlights
Me2SO1 is a polar solvent used to promote cell differentiation of tumor cell lines
Me2SO Affects Alternative Pre-mRNA Splicing in Vivo— Me2SO can promote cell differentiation, a process that is often associated with a change in the alternative splicing profile of specific genes
We have observed that the addition of Me2SO to nuclear extracts can have strong effects on splice site selection while having minimal effects on the efficiency of splicing
Summary
Me2SO, dimethyl sulfoxide; DMF, N,Ndimethylformamide; nt, nucleotide(s); bp, base pairs; hnRNP, heterogeneous nuclear ribonucleoprotein; NCAM, neural cell adhesion molecule; GST, glutathione S-transferase; PCR, polymerase chain reaction. The cellular mechanisms that are affected by Me2SO remain unclear. Me2SO treatment promotes changes in the abundance of certain mRNAs and in the ratio of spliced isoforms (14 –17). Among the genes reported to be affected in their alternative splicing is the NCAM pre-mRNA. Me2SO alters the alternative splicing of other genes including the amyloid precursor protein [20], the serotonin 5-HT3 receptor-A mRNA [21], and p53 [22, 23]. Because treating cells with Me2SO can have a strong effect on the alternative splicing of many pre-mRNAs and because the mechanism of action of Me2SO remains unclear, we performed a series of experiments in nuclear extracts to assess whether Me2SO directly affects the activity of the splicing machinery. Other solvents of the same category (e.g. DMF and formamide) perturb splice site selection
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