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RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes 1 , 2 . Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex 1 – 5 . Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein 6 —is phosphorylated during spliceosome activation 7 – 10 , but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing. CDK11 associates with SF3B1 and phosphorylates threonine residues at the N terminus of SF3B1 during spliceosome activation, and the inhibition of CDK11 blocks the activation and leads to widespread intron retention and the accumulation of non-functional spliceosomes on pre-mRNAs and chromatin.

Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy. Arginine methyltransferases (PRMTs) are involved in the regulation of various physiological and pathological conditions. Using proteomics, the authors here profile the methylation substrates of PRMTs 4, 5 and 7 and characterize the roles of these enzymes in cancer-associated splicing regulation.

Splicing factors such as BUD31 are identified in a synthetic-lethal screen with cells overexpressing the transcription factor MYC; oncogenic MYC leads to an increase in pre-mRNA synthesis, and spliceosome inhibition impairs the growth and tumorigenicity of MYC-dependent breast cancers, suggesting that spliceosome components may be potential therapeutic targets for MYC-driven cancers. Tolerating overexpressed MYC The transcription factor MYC is frequently amplified or overexpressed in cancer and drives increased RNA and protein production. Here, Thomas Westbrook and colleagues identify the splicing factor BUD31 in a synthetic lethal screen with cells overexpressing MYC and show that other splicing factors are also required for cells to tolerate overexpressed MYC. Oncogenic MYC leads to an increase in pre-mRNA synthesis, and inhibition of the spliceosome impairs the growth and tumorigenicity of MYC-dependent breast cancer cells. Spliceosome components may therefore be potential therapeutic targets for MYC-driven cancers. MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs 1 , 2 , 3 . Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts 4 , 5 , 6 , 7 . While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly 8 . Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures. Small molecules correcting the splicing deficit of the survival of motor neuron 2 ( SMN2 ) gene have been identified as having therapeutic potential. Here, the authors provide evidence that SMN2 mRNA forms a ribonucleoprotein complex that can be specifically targeted by these small molecules.

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers. Splicing is a vital biological reaction and a druggable pathway to treat infection, genetic diseases and cancer. Here, the authors describe how splicing is modulated by small molecules that target the conserved splicing active site in the group II introns.

Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma. The mechanisms of action of proteasome inhibitors (PI) in multiple myeloma (MM) treatment are not fully elucidated. Here, the authors use unbiased phosphoproteomics in PI-treated MM and show increased phosphorylation of splicing-associated proteins, ultimately revealing splicing interference as a mode of PI action as well as demonstrating the spliceosome as a specific therapeutic vulnerability in this disease.

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1 , BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL ( BCL2L1 -encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment. Small molecule modulators of RNA splicing have therapeutic potential in tumours bearing spliceosome mutations. Here, the authors identify BCL2 genes have differential sensitivities to SF3b-targeting splicing modulators and combination of SF3b-targeting splicing modulators and BCLxL inhibition induces synergistic cytotoxicity in cancer cells.

There has been accumulating evidence that RNA splicing is frequently dysregulated in a variety of cancers and that hotspot mutations affecting key splicing factors, SF3B1, SRSF2 and U2AF1, are commonly enriched across cancers, strongly suggesting that aberrant RNA splicing is a new class of hallmark that contributes to the initiation and/or maintenance of cancers. In parallel, some studies have demonstrated that cancer cells with global splicing alterations are dependent on the transcriptional products derived from wild‐type spliceosome for their survival, which potentially creates a therapeutic vulnerability in cancers with a mutant spliceosome. It has been c. 10 y since the frequent mutations affecting splicing factors were reported in cancers. Based on these surprising findings, there has been a growing interest in targeting altered splicing in the treatment of cancers, which has promoted a wide variety of investigations including genetic, molecular and biological studies addressing how altered splicing promotes oncogenesis and how cancers bearing alterations in splicing can be targeted therapeutically. In this mini‐review we present a concise trajectory of what has been elucidated regarding the pathogenesis of cancers with aberrant splicing, as well as the development of therapeutic strategies to target global splicing alterations in cancers. Transcription and pre‐mRNA splicing are key steps in the control of gene expression in eukaryotic cells and mutations in genes regulating each of these processes are common in cancers. By reviewing the recent advances in this field, we described the pathogenic mechanisms in which the hotspot mutations in genes encoding splicing factors drive oncogenesis, and therapeutic strategies for targeting global alterations in splicing, including the use of splicing modulators, inhibition of splicing regulatory proteins, emerging technologies using antisense oligonucleotides and a potential tactic to improve the response to checkpoint blockade.

Despite the importance of spliceosome core components in cellular processes, their roles in cancer development, including hepatocellular carcinoma (HCC), remain poorly understood. In this study, we uncover a critical role for SmD2, a core component of the spliceosome machinery, in modulating DNA damage in HCC through its impact on BRCA1 / FANC cassette exons and expression. Our findings reveal that SmD2 depletion sensitizes HCC cells to PARP inhibitors, expanding the potential therapeutic targets. We also demonstrate that SmD2 acetylation by p300 leads to its degradation, while HDAC2-mediated deacetylation stabilizes SmD2. Importantly, we show that the combination of Romidepsin and Olaparib exhibits significant therapeutic potential in multiple HCC models, highlighting the promise of targeting SmD2 acetylation and HDAC2 inhibition alongside PARP inhibitors for HCC treatment. The relevance of spliceosome core components in cancer is less understood. Here the authors show SmD2, a core component of the spliceosome machinery, is under acetylation-dependent regulation, which could be targeted to enhance sensitivity to PARP inhibitors in hepatocellular carcinoma.

Significance Alternative splicing of RNA allows a limited number of coding regions in the human genome to produce proteins with diverse functionality. Alternative splicing has also been implicated as an oncogenic process. Identifying aspects of cancer cells that differentiate them from noncancer cells remains an ongoing challenge, and our research suggests that alternatively spliced mRNA and subsequent protein isoforms will provide new anticancer targets. We determined that the key oncoprotein of Ewing sarcoma (ES), EWS-FLI1, regulates alternative splicing in multiple cell line models. These experiments establish oncogenic aspects of splicing that are specific to cancer cells and thereby illuminate potentially oncogenic splicing shifts as well as provide a useful stratification mechanism for ES patients. The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1 , CASP3 , PPFIBP1 , and TERT , validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.