Functions of Replication Protein A as a Sensor of R Loops and a Regulator of RNaseH1.

R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here we show that replication protein A (RPA), an ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.

Pre-mRNA splicing-associated diseases and therapies.

Precursor mRNA (pre-mRNA) splicing is an essential step in human gene expression and is carried out by a large macromolecular machine called the spliceosome. Given the spliceosome's role in shaping the cellular transcriptome, it is not surprising that mutations in the splicing machinery can result in a range of human diseases and disorders (spliceosomopathies). This review serves as an introduction into the main features of the pre-mRNA splicing machinery in humans and how changes in the function of its components can lead to diseases ranging from blindness to cancers. Recently, several drugs have been developed that interact directly with this machinery to change splicing outcomes at either the single gene or transcriptome-scale. We discuss the mechanism of action of several drugs that perturb splicing in unique ways. Finally, we speculate on what the future may hold in the emerging area of spliceosomopathies and spliceosome-targeted treatments.

Apoptosis induction and cell cycle arrest of pladienolide B in erythroleukemia cell lines.

Splicing of pre-mRNA into functional mRNA, carried out by the spliceosome, represents a crucial step in eukaryotic gene expression. Mutations and other deregulation in some of the spliceosome components have been identified in multiple pathologies, including hematological malignancies. In this context, we evaluated the therapeutic potential of a splicing inhibitor, Pladienolide B (Pla-B), in two erythroleukemia cell lines. HEL and K562 cell lines were incubated with increasing doses of Pla-B in single and daily administration. Cell viability and density were evaluated using trypan blue assay. Flow cytometry was used to evaluate cell death, cell cycle, and caspase activity. NGS analysis was performed to assess the mutational status of 4 splicing-related genes (SF3B1, U2AF1, ZRSR2 and SRSF2). Expression levels of SF3B1 and unspliced DNAJB1 were evaluated by qPCR. Pla-B significantly decreased the viability and proliferation of both cell lines in time, dose, administration schedule, and cell line-dependent manner. HEL cells were more sensible to Pla-B (IC50 = 1.5 nM) than K562 (IC50 = 25 nM), with an IC50 almost 17 times lower. Pla-B induced cell death, mainly by apoptosis, and cell cycle arrest in G0/G1 phase. No mutations were found in any of the analyzed genes, suggesting that the observed cytotoxic effect is independent of the spliceosome mutations. Splicing modulator Pla-B showed high antitumor activity against HEL and K562 cell lines, inducing apoptosis and cell cycle arrest. These data suggest that Pla-B might represent a new therapeutic approach for erythroleukemia.

Regulating Divergent Transcriptomes through mRNA Splicing and Its Modulation Using Various Small Compounds.

Human transcriptomes are more divergent than genes and contribute to the sophistication of life. This divergence is derived from various isoforms arising from alternative splicing. In addition, alternative splicing regulated by spliceosomal factors and RNA structures, such as the RNA G-quadruplex, is important not only for isoform diversity but also for regulating gene expression. Therefore, abnormal splicing leads to serious diseases such as cancer and neurodegenerative disorders. In the first part of this review, we describe the regulation of divergent transcriptomes using alternative mRNA splicing. In the second part, we present the relationship between the disruption of splicing and diseases. Recently, various compounds with splicing inhibitor activity were established. These splicing inhibitors are recognized as a biological tool to investigate the molecular mechanism of splicing and as a potential therapeutic agent for cancer treatment. Food-derived compounds with similar functions were found and are expected to exhibit anticancer effects. In the final part, we describe the compounds that modulate the messenger RNA (mRNA) splicing process and their availability for basic research and future clinical potential.

Biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp.

A key challenge in natural products drug discovery is compound supply. Hundreds of grams of purified material are needed to advance a natural product lead through preclinical development. Spliceostatins are polyketide-nonribosomal peptide natural products that bind to the spliceosome, an emerging target in cancer therapy. The wild-type bacterium Burkholderia sp. FERM BP-3421 produces a suite of spliceostatin congeners with varying biological activities and physiological stabilities. Hemiketal compounds such as FR901464 were the first to be described. Due to its improved properties, we were particularly interested in a carboxylic acid precursor analog that was first reported from Burkholderia sp. MSMB 43 and termed thailanstatin A. Inactivation of the iron/α-ketoglutarate-dependent dioxygenase gene fr9P had been shown to block hemiketal biosynthesis. However, a 4-deoxy congener of thailanstatin A was the main product seen in the dioxygenase mutant. We show here that expression of the cytochrome P450 gene fr9R is a metabolic bottle neck, as use of an l-arabinose inducible system led to nearly complete conversion of the 4-deoxy analog to the target molecule. By integrating fermentation media development approaches with biosynthetic engineering, we were able to improve production titers of the target compound >40-fold, going from the starting ~60 mg/L to 2.5 g/L, and to achieve what is predominantly a single component production profile. These improvements were instrumental in enabling preclinical development of spliceostatin analogs as chemotherapy.

Defective splicing of the background K+ channel K2P5.1 by the pre-mRNA splicing inhibitor, pladienolide B in lectin-activated mouse splenic CD4+ T cells.

The two-pore domain K+ channel K2P5.1 has been implicated in the pathogenesis of autoimmune diseases. We investigated the changes in K2P5.1 activity caused by a defect in normal pre-mRNA splicing in concanavalin A-activated mouse splenic CD4+ T cells. The pre-mRNA splicing inhibitor, pladienolide B (1 µM) markedly decreased full-length K2P5.1 transcription in activated CD4+ T cells, resulting in the disappearance of K2P5.1 activity and an imbalance in Th17 and Treg cytokines. These results suggest that the defect in K2P5.1 splicing by the pre-mRNA splicing inhibitor regulates pro- and/or anti-inflammatory cytokine production in K2P5.1-associated autoimmune diseases.

Identification of a small molecule splicing inhibitor targeting UHM domains.

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.

Identification of a novel and potent small molecule inhibitor of SRPK1: mechanism of dual inhibition of SRPK1 for the inhibition of cancer progression.

Protein kinases are the family of attractive enzyme targets for drug design with relevance to cancer biology. Serine arginine protein kinase 1 (SRPK1) is responsible for the phosphorylation of serine/arginine (SR)-rich proteins. Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2) involved in mRNA editing. ASF/SF2 is over expressed in many cancers and plays crucial roles in the cell survival. Phosphorylation of ASF/SF2 is decisive for its functions in cancer. In search of potential anticancer therapeutic agents for attenuating phosphorylation of ASF/SF2, we have explored specific and potential inhibitors of SRPK1 from natural and drug like compounds databases using in-silico methods. Compound ZINC02154892 (C02) was found to be the most potent inhibitor for SRPK1. In-vitro molecular and cell biology studies have shown C02 as a potent and specific inhibitor of phosphorylation of ASF/SF2 and cell survival in leukemic cell line. Structural analysis of SRPK1 with compound C02 revealed a unique pattern of binding targeting ATP binding site along with inhibiting recruitment of ASF/SF2 by SRPK1. The possibilities of compound C02 to be used as a lead compound paving way for the development of potent and specific inhibitors of SRPK1 for designing of novel potential anticancer inhibitor is inferred from the current studies.