Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3′-end processing and associated splicing factors.

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg2+/Ca2+-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.

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Periodic accumulation and degradation of RAG2 (recombination-activating gene 2) protein controls the cellcycle-dependent V(D)J recombination of lymphocyte antigen receptor genes. Here we show the molecular mechanism of RAG2 degradation. The RAG2 protein is translocated from the nucleus to the cytoplasm and degraded through the ubiquitin/proteasome system. RAG2 translocation is mediated by the Thr-490 phosphorylation of RAG2. Inhibition of this phosphorylation by p27Kip1 stabilizes the RAG2 protein in the nucleus. These results suggest that RAG2 sequestration in the cytoplasm and its subsequent degradation by the ubiquitin/proteasome system upon entering the S phase is an integral part of G0/G1-specific V(D)J recombination.During B and T cell development, the genes encoding the variable region of immunoglobulin (Ig) and the T cell receptor (TCR) are assembled from germ line-variable (V), diversity (D), and joining (J) gene segments by V(D)J recombination (1-3). V(D)J recombination is initiated by the introduction of sitespecific double-strand breaks (DSBs) between two recombining gene segments and their flanking recombination signal sequences (RSSs). The essential components of this reaction are RAG1 and RAG2 (recombination-activating genes 1 and 2) proteins, which are expressed specifically in the developing B and T lineage cells under V(D)J recombination (4, 5). Subsequent reaction steps employ more generally expressed proteins involved in non-homologous end joining (6, 7): the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the Ku70/80 dimer, XRCC4, and DNA Ligase IV. More proteins may be involved in V(D)J recombination to ensure the strict regulation of this system. Several lines of evidence suggest that V(D)J recombination is restricted to the G0/G1 stage of the cell cycle and that periodic accumulation and destruction of the RAG2 protein regulate cell-cycle-dependent V(D)J recombination (8). According to Lee and Desiderio, Thr-490 of RAG2 is phosphorylated by cyclin A/CDK2 when the cells are at the G1-S transition of the cell cycle, thereby triggering the rapid degradation of RAG2 (9). However, the precise molecular mechanism of the degradation process remains elusive.The ubiquitin/proteasome system plays a major role in target-specific protein degradation and in the regulation of protein expression levels (10, 11). The formation of ubiquitin-protein conjugates proceeds via a three-step cascade. First, a ubiquitinactivating enzyme (E1) activates ubiquitin, which is then transferred by a ubiquitin-conjugating enzyme (E2) to a ubiquitin ligase (E3) with which the substrate protein is associated. Finally, E3 catalyzes the conjugation of ubiquitin to the substrate protein. Proteins polyubiquitinated by these enzymes are subjected to degradation by the 26S proteasome. Recent reports have suggested that many proteins, such as p53, IB, ␤-catenin, and p27Kip1, are degraded by the ubiquitin/proteasome pathway (10 -13).p27Kip1, a cyclin-dependent kinase inhibitor, increases RAG2 stability by inhibiting ...

The RNA helicase EIF4A3 regulates the exon junction complex and nonsense-mediated mRNA decay functions in RNA transcript processing. However, a transcriptome-wide network definition of these functions has been lacking, in part due to the lack of suitable pharmacological inhibitors. Here we employ short-duration graded EIF4A3 inhibition using small molecule allosteric inhibitors to define the transcriptome-wide dependencies of EIF4A3. We thus define conserved cellular functions, such as cell cycle control, that are EIF4A3 dependent. We show that EIF4A3-dependent splicing reactions have a distinct genome-wide pattern of associated RNA-binding protein motifs. We also uncover an unanticipated role of EIF4A3 in the biology of RNA stress granules, which sequester and silence the translation of most mRNAs under stress conditions and are implicated in cell survival and tumour progression. We show that stress granule induction and maintenance is suppressed on the inhibition of EIF4A3, in part through EIF4A3-associated regulation of G3BP1 and TIA1 scaffold protein expression.

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Cyclin-dependent kinase 12 (CDK12) plays a key role in the coordination of transcription with elongation and mRNA processing. CDK12 mutations found in tumors and CDK12 inhibition sensitize cancer cells to DNA-damaging reagents and DNA-repair inhibitors. This suggests that CDK12 inhibitors are potential therapeutics for cancer that may cause synthetic lethality. Here, we report the discovery of 3-benzyl-1-( trans-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-arylurea derivatives as novel and selective CDK12 inhibitors. Structure-activity relationship studies of a HTS hit, structure-based drug design, and conformation-oriented design using the Cambridge Structural Database afforded the optimized compound 2, which exhibited not only potent CDK12 (and CDK13) inhibitory activity and excellent selectivity but also good physicochemical properties. Furthermore, 2 inhibited the phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and induced growth inhibition in SK-BR-3 cells. Therefore, 2 represents an excellent chemical probe for functional studies of CDK12 and could be a promising lead compound for drug discovery.