A model for DHX15 mediated disassembly of A-complex spliceosomes.

A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation, and several RNA-dependent ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement, and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNP's engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 may have an expanded role in maintaining splicing fidelity.

U2 snRNA structure is influenced by SF3A and SF3B proteins but not by SF3B inhibitors.

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP's association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5' end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

Spliceostatins and Derivatives: Chemical Syntheses and Biological Properties of Potent Splicing Inhibitors.

Spliceostatins and thailanstatins are intriguing natural products due to their structural features as well as their biological significance. This family of natural products has been the subject of immense synthetic interest because they exhibit very potent cytotoxicity in representative human cancer cell lines. The cytotoxic properties of these natural products are related to their ability to inhibit spliceosomes. FR901564 and spliceostatins have been shown to inhibit spliceosomes by binding to their SF3B component. Structurally, these natural products contain two highly functionalized tetrahydropyran rings with multiple stereogenic centers joined by a diene moiety and an acyclic side chain linked with an amide bond. Total syntheses of this family of natural products led to the development of useful synthetic strategies, which enabled the synthesis of potent derivatives. The spliceosome modulating properties of spliceostatins and synthetic derivatives opened the door for understanding the underlying spliceosome mechanism as well as the development of new therapies based upon small-molecule splicing modulators. This review outlines the total synthesis of spliceostatins, synthetic studies of structural derivatives, and their bioactivity.

A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex.

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.

Herboxidiene Features That Mediate Conformation-Dependent SF3B1 Interactions to Inhibit Splicing.

Small molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Employing an inactive herboxidiene analog (iHB) as a competitor, we investigated factors that influence inhibitor interactions with SF3B to interfere with pre-mRNA splicing in vitro. Order-of-addition experiments show that inhibitor interactions are long lasting and affected by both temperature and the presence of ATP. Our data are also consistent with the model that not all SF3B conformations observed in structural studies are conducive to productive inhibitor interactions. Notably, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure-activity relationship analysis of the splicing inhibitor herboxidiene. We identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In the context of recent structures of SF3B bound to inhibitor, our results lead us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 side chains in the inhibitor binding channel are needed to maintain inhibitor occupancy while counteracting the SF3B transition to a closed state that is required for stable U2 snRNP interactions with the intron.

Design and synthesis of herboxidiene derivatives that potently inhibit in vitro splicing.

Herboxidiene is a potent antitumor agent that targets the SF3B subunit of the spliceosome. Herboxidiene possesses a complex structural architecture with nine stereocenters and design of potent less complex structures would be of interest as a drug lead as well as a tool for studying SF3B1 function in splicing. We investigated a number of C-6 modified herboxidiene derivatives in an effort to eliminate this stereocenter and, also to understand the importance of this functionality. The syntheses of structural variants involved a Suzuki-Miyaura cross-coupling reaction as the key step. The functionalized tetrahydrofuran core has been constructed from commercially available optically active tri-O-acetyl-d-glucal. We investigated the effect of these derivatives on splicing chemistry. The C-6 alkene derivative showed very potent splicing inhibitory activity similar to herboxidiene. Furthermore, the C-6 gem-dimethyl derivative also exhibited very potent in vitro splicing inhibitory activity comparable to herboxidiene.

Pharmacological Targeting of Vacuolar H+-ATPase via Subunit V1G Combats Multidrug-Resistant Cancer.

Multidrug resistance (MDR) in cancer remains a major challenge for the success of chemotherapy. Natural products have been a rich source for the discovery of drugs against MDR cancers. Here, we applied high-throughput cytotoxicity screening of an in-house natural product library against MDR SGC7901/VCR cells and identified that the cyclodepsipeptide verucopeptin demonstrated notable antitumor potency. Cytological profiling combined with click chemistry-based proteomics revealed that ATP6V1G directly interacted with verucopeptin. ATP6V1G, a subunit of the vacuolar H+-ATPase (v-ATPase) that has not been previously targeted, was essential for SGC7901/VCR cell growth. Verucopeptin exhibited strong inhibition of both v-ATPase activity and mTORC1 signaling, leading to substantial pharmacological efficacy against SGC7901/VCR cell proliferation and tumor growth in vivo. Our results demonstrate that targeting v-ATPase via its V1G subunit constitutes a unique approach for modulating v-ATPase and mTORC1 signaling with great potential for the development of therapeutics against MDR cancers.

Copper-Catalyzed Stille Cross-Coupling Reaction and Application in the Synthesis of the Spliceostatin Core Structure.

An efficient palladium-free Stille cross-coupling reaction of allylic bromides and functionalized organostannylfuran using catalytic copper halide has been developed. The coupling reaction was optimized using CuI and low catalyst loading (down to 5 mol %). The reaction was conveniently carried out at ambient temperature in the presence of inorganic base to afford the coupling product in good-to-excellent yields. The utility of this reaction was demonstrated in the synthesis of a furan with sensitive functionalities. A sulfolene moiety was utilized as a masking group for the sensitive diene. Noyori asymmetric reduction, Achmatowicz reaction, and Kishi reduction steps converted sulfolene to a highly substituted tetrahydropyran intermediate used in the synthesis of the highly potent antitumor agents, spliceostatins, and their derivatives.

A two-step probing method to compare lysine accessibility across macromolecular complex conformations.

Structural models of large and dynamic molecular complexes are appearing in increasing numbers, in large part because of recent technical advances in cryo-electron microscopy. However, the inherent complexity of such biological assemblies comprising dozens of moving parts often limits the resolution of structural models and leaves the puzzle as to how each functional configuration transitions to the next. Orthogonal biochemical information is crucial to understanding the molecular interactions that drive those rearrangements. We present a two-step method for chemical probing detected by tandem mass-spectrometry to globally assess the reactivity of lysine residues within purified macromolecular complexes. Because lysine side chains often balance the negative charge of RNA in ribonucleoprotein complexes, the method is especially useful for detecting changes in protein-RNA interactions. By probing the E. coli 30S ribosome subunit, we established that the reactivity pattern of lysine residues quantitatively reflects structure models derived from X-ray crystallography. We also used the strategy to assess differences in three conformations of purified human spliceosomes in the context of recent cryo-electron microscopy models. Our results demonstrate that the probing method yields powerful biochemical information that helps contextualize architectural rearrangements of intermediate resolution structures of macromolecular complexes, often solved in multiple conformations.

Human RNF113A participates of pre-mRNA splicing in vitro.

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Human RNF113A participates of pre-mRNA splicing in vitro

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Human RNF113A participates of pre-mRNA splicing in vitro

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Enantioselective Synthesis of a Cyclopropane Derivative of Spliceostatin A and Evaluation of Bioactivity.

Spliceostatin A is a potent inhibitor of spliceosomes and exhibits excellent anticancer activity against multiple human cancer cell lines. We describe here the design and synthesis of a stable cyclopropane derivative of spliceostatin A. The synthesis involved a cross-metathesis or a Suzuki cross-coupling reaction as the key step. The functionalized epoxy alcohol ring was constructed from commercially available optically active tri- O-acetyl-d-glucal. The biological properties of the cyclopropyl derivative revealed that it is active in human cells and inhibits splicing in vitro comparable to spliceostatin A.

Enantioselective Synthesis of Thailanstatin A Methyl Ester and Evaluation of in Vitro Splicing Inhibition.

Thailanstatin A has been isolated recently from the fermentation broth of B. thailandensis MSMB43. We describe here an enantioselective convergent synthesis of thailanstatin A methyl ester and evaluation of its splicing activity. Synthesis of both highly functionalized tetrahydropyran rings were carried out from commercially available tri- O-acetyl-d-glucal as the key starting material. Our convergent synthesis involved the synthesis of both tetrahydropyran fragments in a highly stereoselective manner. The fragments were then coupled using cross-metathesis as the key step. The synthesis of the diene subunit included a highly stereoselective Claisen rearrangement, a Cu(I)-mediated conjugate addition of MeLi to set the C-14 methyl stereochemistry, a reductive amination reaction to install the C16-amine functionality, and a Wittig olefination reaction to incorporate the diene unit. The epoxy alcohol subunit was synthesized by a highly selective anomeric allylation, a Peterson olefination, and a vanadium catalyzed epoxidation that installed the epoxide stereoselectively. Cross-metathesis of the olefins provided the methyl ester derivative of thailanstatin A. We have carried out in vitro splicing studies of the methyl ester derivative, which proved to be a potent inhibitor of the spliceosome.

Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.

Prp8 is an essential protein that regulates spliceosome assembly and conformation during pre-mRNA splicing. Recent cryo-EM structures of the spliceosome model Prp8 as a scaffold for the spliceosome's catalytic U snRNA components. Using a new amino acid probing strategy, we identified a dynamic region in human Prp8 that is positioned to stabilize the pre-mRNA in the spliceosome active site through interactions with U5 snRNA. Mutagenesis of the identified Prp8 residues in yeast indicates a role in 5' splice site recognition. Genetic interactions with spliceosome proteins Isy1, which buttresses the intron branch point, and Snu114, a regulatory GTPase that directly contacts Prp8, further corroborate a role for the same Prp8 residues in substrate positioning and activation. Together the data suggest that adjustments in interactions between Prp8 and U5 snRNA help establish proper positioning of the pre-mRNA into the active site to enhance 5' splice site fidelity.

Enantioselective Synthesis of Spliceostatin G and Evaluation of Bioactivity of Spliceostatin G and Its Methyl Ester.

An enantioselective total synthesis of spliceostatin G has been accomplished. The synthesis involved a Suzuki cross-coupling reaction as a key step. The functionalized tetrahydropyran ring was constructed from commercially available optically active tri-O-acetyl-d-glucal. Other key reactions include a highly stereoselective Claisen rearrangement, a Cu(I)-mediated 1,4 addition of MeLi to install the C8 methyl group, and a reductive amination to incorporate the C10 amine functionality of spliceostatin G. Biological evaluation of synthetic spliceostatin G and its methyl ester revealed that it does not inhibit splicing in vitro.

Modulating splicing with small molecular inhibitors of the spliceosome.

Small molecule inhibitors that target components of the spliceosome have great potential as tools to probe splicing mechanism and dissect splicing regulatory networks in cells. These compounds also hold promise as drug leads for diseases in which splicing regulation plays a critical role, including many cancers. Because the spliceosome is a complicated and dynamic macromolecular machine comprised of many RNA and protein components, a variety of compounds that interfere with different aspects of spliceosome assembly is needed to probe its function. By screening chemical libraries with high-throughput splicing assays, several labs have added to the collection of splicing inhibitors, although the mechanistic insight into splicing yielded from the initial compound hits is somewhat limited so far. In contrast, SF3B1 inhibitors stand out as a great example of what can be accomplished with small molecule tools. This group of compounds were first discovered as natural products that are cytotoxic to cancer cells, and then later shown to target the core spliceosome protein SF3B1. The inhibitors have since been used to uncover details of SF3B1 mechanism in the spliceosome and its impact on gene expression in cells. Continuing structure activity relationship analysis of the compounds is also making progress in identifying chemical features key to their function, which is critical in understanding the mechanism of SF3B1 inhibition. The knowledge is also important for the design of analogs with new and useful features for both splicing researchers and clinicians hoping to exploit splicing as pressure point to target in cancer therapy. WIREs RNA 2017, 8:e1381. doi: 10.1002/wrna.1381 For further resources related to this article, please visit the WIREs website.

Design, synthesis and in vitro splicing inhibition of desmethyl and carba-derivatives of herboxidiene.

Herboxidiene is a potent inhibitor of spliceosomes. It exhibits excellent anticancer activity against multiple human cancer cell lines. Herein, we describe an enantioselective synthesis of a desmethyl derivative and the corresponding carba-derivatives of herboxidiene. The synthesis involved Suzuki coupling of a vinyl iodide with boronate as the key reaction. For the synthesis of carba-derivatives, the corresponding optically active cyclohexane-1,3-dicarbonyl derivatives were synthesized using an enantioselective desymmetrization of meso-anhydride. The biological properties of these derivatives were evaluated in an in vitro splicing assay.

Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages.

The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1's role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.

The Natural Product N-Palmitoyl-l-leucine Selectively Inhibits Late Assembly of Human Spliceosomes.

The spliceosome is a dynamic complex of five structural RNAs and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks, we identified N-palmitoyl-l-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure-activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function.