Strukturelle Untersuchungen der Spleißregulation unter pathologischen und Komponenten-induzierten Bedingungen
Zusammenfassung der Projektergebnisse
Compounds that bind the SF3B complex and modulate splicing (i.e., splicing modulators) were identified over two decades ago. Since then, different labs have extensively studied and developed them, given their therapeutic potential as anti-cancer drugs. Yet, their molecular recognition and mechanism of action were unknown in structural terms before this study. Besides, the reason why they stalled prespliceosomes was largely unclear. The funding from this proposal enabled us to describe the crystal structures of the recombinant SF3B complex bound to several splicing modulators. The structures showed that these compounds act as competitive antagonists of branch sites during the formation of the prespliceosomes, and enabled a thorough description of their molecular recognition. Furthermore, our structural analysis revealed that spliceostatin and sudemycin belang to a distinct class of SF3B ligands that covalently couple to a zinc finger within prespliceosomes. W e identified the warhead group of these compounds and elucidated the underlying reaction mechanism governing their coupling. This knowledge can be leveraged to design more potent and effective drugs targeting SF3B. Along a second line of investigation, we employed spliceostatin as a molecular tool to stall human prespliceosomes, enabling us to dissect unknown structural transitions of the splicing pathway. This approach facilitated the isolation and cryo-EM structure determination of a core complex found midway between E and A prespliceosomes. This intermediate unveiled unexpected transitions in the prespliceosome formation process, where introns are initially bound through a precursor duplex with U2 snRNA before incorporating the branch site adenosine. This finding shed light on the selection of introns by U2 snRNP through a novel mechanism involving toehold-mediated strand displacement, which had not been observed previously in pre-mRNA splicing. Lastly, the structural information acquired from our research offers a plausible explanation for the sequence-dependent inactivation of introns induced by these anti-cancer compounds. Part of these results were published in Molecular Cell, while the most significant findings were later reported in Nature Communications.
Projektbezogene Publikationen (Auswahl)
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Structural Basis of Splicing Modulation by Antitumor Macrolide Compounds. Molecular Cell, 70(2), 265-273.e8.
Cretu, Constantin; Agrawal, Anant A.; Cook, Andrew; Will, Cindy L.; Fekkes, Peter; Smith, Peter G.; Lührmann, Reinhard; Larsen, Nicholas; Buonamici, Silvia & Pena, Vladimir
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Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors. Nature Communications, 12(1).
Cretu, Constantin; Gee, Patricia; Liu, Xiang; Agrawal, Anant; Nguyen, Tuong-Vi; Ghosh, Arun K.; Cook, Andrew; Jurica, Melissa; Larsen, Nicholas A. & Pena, Vladimir
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Structural basis of catalytic activation in human splicing. Nature, 617(7962), 842-850.
Schmitzová, Jana; Cretu, Constantin; Dienemann, Christian; Urlaub, Henning & Pena, Vladimir
