Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors

Abstract

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)—a complex chaperoning the selection of branch and 3′ splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.

Structural basis of branch site recognition by the human spliceosome

“…Rejection of weak, suboptimal substrates results in the remodeled U2 snRNP, which is targeted to a discard pathway (this work). Stable substrates gradually form the branch helix as shown in the E-to-A ( 41 ) and pre-A ( 42 ) intermediates. In the absence of properly positioned, bulged out BP-A, the pre-A complex is targeted to a discard pathway.…”

“…BS sequences that withstand competition with the BMSL would continue to progressively form the branch helix through a recently proposed toehold strand invasion mechanism ( 41 ). An intermediate state in this process (A3′-SSA complex) was captured by blocking spliceosome assembly with spliceostatin A (SSA) ( 41 ), which trapped U2 snRNP with a partially formed branch helix, missing bulged out BP-A. Consequently, the branch helix was not accommodated in its pocket and SF3B1 remains in the open conformation, resembling that found in the 17 S U2 snRNP (Fig.…”

Investigating the Molecular Mechanism of H3B-8800: A Splicing Modulator Inducing Preferential Lethality in Spliceosome-Mutant Cancers

“…The most recurrent K700E SF3B1 mutation often occurs in patients affected by myelodysplastic syndromes, such as myelodysplasia with ring sideroblasts [13]. In this scenario, the SPL is emerging as an appealing target for cancer treatment via small molecules, also due to the increased understanding of its mechanism provided by recent structural [14][15][16], functional [17,18], and computational studies [19,20]. SF3b was the first SF for which structural information in complex with small-molecules splicing modulators (SMs) were solved [14][15][16].…”

“…In this scenario, the SPL is emerging as an appealing target for cancer treatment via small molecules, also due to the increased understanding of its mechanism provided by recent structural [14][15][16], functional [17,18], and computational studies [19,20]. SF3b was the first SF for which structural information in complex with small-molecules splicing modulators (SMs) were solved [14][15][16]. These SMs were initially discovered from natural products and later derivatized in drug discovery studies.…”

Somatic Mutations in Core Spliceosome Components Promote Tumorigenesis and Generate an Exploitable Vulnerability in Human Cancer

“…Interestingly, although structurally similar, SSA and Sudemycins differentially affected splicing of target genes. Both FR901464 analogues act at the latest stages of intron selection by the U2 snRNP by “locking” the SF3B1 subunit in an open conformation and preventing the formation of the extended U2/intron duplex [ 85 ]. However, sudemycins preferentially regulate exon skipping events, whereas SSA displays stronger effects on intron retention [ 62 ].…”