Abstract
Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2) and a molecule currently in clinical trials (Phase II) for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks) in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR) proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3–90 nM) was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.
Highlights
The removal of introns and rejoining of adjacent exons from nascent transcripts by the process of pre-mRNA splicing is an essential step in eukaryotic gene expression [1]
In our preliminary experiments that investigated a possible role of casein kinase 2 (CK2) in other physiologies including lipid metabolism, we occasionally observed that CX-4945, a wellcharacterized potent inhibitor of CK2, induced alterations in the splicing pattern of CK2 a9 pre-mRNA
CK2 a9 gexon6 protein was barely detectable in several cultured cell types even after CX-4945 treatment. These results demonstrate that CX-4945 induces an abnormal alternative splicing of CK2 a9 pre-mRNA and suggest that CX-4945 has an effect on pre-mRNA splicing
Summary
The removal of introns and rejoining of adjacent exons from nascent transcripts by the process of pre-mRNA splicing is an essential step in eukaryotic gene expression [1]. Most pre-mRNAs in higher eukaryotes can be spliced in several different ways to produce multiple mRNAs in a process called alternative splicing, allowing a single gene sequence to be expressed as numerous protein isoforms with different functions [2]. In this way, alternative splicing contributes to the cellular complexity and generates the phenotypic diversity of higher eukaryotes without the need to expand the genome [3]. Aberrations in splicing due to mutations in pre-mRNAs or splicing machinery have been increasingly found to be associated with a wide range of human diseases, such as cancers, neurodegenerative diseases, viral diseases, and autoimmune diseases [3,6,7,8,9]
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