Breaking Up the C Complex Spliceosome Shows Stable Association of Proteins with the Lariat Intron Intermediate

Spliceosome assembly requires several structural rearrangements to position the components of the catalytic core. Many of these rearrangements involve successive strengthening and weakening of different RNA∶RNA and RNA∶proteins interactions within the complex. To gain insight into the organization of the catalytic core of the spliceosome arrested between the two steps of splicing chemistry (C complex), we investigated the effects of exposing C complex to low concentrations of urea. We find that in the presence of 3M urea C complex separates into at least three sub-complexes. One sub-complex contains the 5′exon, another contains the intron-lariat intermediate, and U2/U5/U6 snRNAs likely comprise a third sub-complex. We purified the intron-lariat intermediate sub-complex and identified several proteins, including U2 snRNP and PRP19 complex (NTC) components. The data from our study indicate that U2 snRNP proteins in C complex are more stably associated with the lariat-intron intermediate than the U2 snRNA. The results also suggest a set of candidate proteins that hold the lariat-intron intermediate together in C complex. This information is critical for further interpreting the complex architecture of the mammalian spliceosome

Dissecting spliceosome function with small-molecule inhibitors

In eukaryotes, a crucial step in gene expression is pre-mRNA splicing by the spliceosome. The spliceosome is a macromolecular machine that removes intervening intron sequences from over 95% of human transcripts to create a functional template for protein synthesis. Precise splicing is essential for correct gene expression, and mutations in the spliceosome are associated with diseases including cancer. Mass spectrometry has identified over 100 spliceosome proteins, but deciphering the function for the vast majority of them has been challenging due to the highly dynamic and complex nature of the spliceosome. My dissertation focuses on small-molecule inhibitors as tools to expand the currently limited mechanistic understanding of the spliceosome: First, I developed a high-throughput assay to rapidly and efficiently screen large compound libraries for small molecules that inhibit splicing. I found three new splicing inhibitors and determined their effect on spliceosome assembly and splicing chemistry. Second, I used synthetic inhibitor analogs for structure-activity studies to identify chemical groups that are responsible for compound activity. I found that the same structural features are required for in vitro and in vivo splicing inhibition, and that the cellular response was mainly due to inhibition of the spliceosome. Third, I used biochemical assays to show that three structurally distinct inhibitors bind to the same site of the spliceosome core protein SF3B1, and that SF3B1 has a functional role throughout the multi-step splicing process. SF3B1 is of particular interest because it is often mutated in cancer, which makes it a promising target for the development of novel chemotherapeutics. My work provides a fast, reliable assay to identify small-molecule splicing inhibitors, a series of in vitro and in vivo assays to characterize their effect on complex assembly, splicing chemistry, and cellular phenotype, and an excellent example of how inhibitors can be utilized to decipher the function of spliceosome proteins. In addition to expanding the mechanistic model of one of the most complex macromolecular machines in the cell, identifying the function of individual spliceosome parts in healthy situations is the necessary first step to determine how changes of these functions in aberrant situations can lead to cancer

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

Total synthesis of GEX1Q1, assignment of C-5 stereoconfiguration and evaluation of spliceosome inhibitory activity.

An enantioselective total synthesis of GEX1Q1 has been accomplished in a convergent manner. The C-5 asymmetric center has now been assigned through synthesis. GEX1Q1 displayed slightly better spliceosome inhibitory activity over its C-5 epimer. The salient features of this synthesis include an asymmetric hetero-Diels-Alder reaction to construct the tetrahydropyran ring and a Suzuki cross-coupling to assemble the key segments

Enantioselective total syntheses of FR901464 and spliceostatin A and evaluation of splicing activity of key derivatives.

FR901464 (1) and spliceostatin A (2) are potent inhibitors of spliceosomes. These compounds have shown remarkable anticancer activity against multiple human cancer cell lines. Herein, we describe efficient, enantioselective syntheses of FR901464, spliceostatin A, six corresponding diastereomers and an evaluation of their splicing activity. Syntheses of spliceostatin A and FR901464 were carried out in the longest linear sequence of 9 and 10 steps, respectively. To construct the highly functionalized tetrahydropyran A-ring, we utilized CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps. The remarkable diastereoselectivity of the Michael addition was specifically demonstrated with different substrates under various reaction conditions. The side chain B was prepared from an optically active alcohol, followed by acetylation and hydrogenation over Lindlar's catalyst. The other densely functionalized tetrahydropyran C-ring was derived from readily available (R)-isopropylidene glyceraldehyde through a route featuring 1,2-addition, cyclic ketalization, and regioselective epoxidation. These fragments were coupled together at a late stage through amidation and cross-metathesis in a convergent manner. Six key diastereomers were then synthesized to probe the importance of specific stereochemical features of FR901464 and spliceostatin A, with respect to their in vitro splicing activity

Total Synthesis of GEX1Q1, Assignment of C‑5 Stereoconfiguration and Evaluation of Spliceosome Inhibitory Activity

An enantioselective total synthesis of GEX1Q1 has been accomplished in a convergent manner. The C-5 asymmetric center has now been assigned through synthesis. GEX1Q1 displayed slightly better spliceosome inhibitory activity over its C-5 epimer. The salient features of this synthesis include an asymmetric hetero-Diels–Alder reaction to construct the tetrahydropyran ring and a Suzuki cross-coupling to assemble the key segments