Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

Abstract

Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudoatomic model of the closed form of SF3b which is found to be a "fuzzy complex" with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5 0 end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity.

A Challenging Pie to Splice: Drugging the Spliceosome

“…[18] Despite the lack of structural data, detailed SARs of the three major families of SPLMs have been determined. [9] Of particular note is work in which four isomers of FD-895 were synthesized.…”

“…[44] While ongoing studies suggest that all three families of natural products ( 1 −− 8 ; Figures 1, 2) share a common binding site within SF3b, a lack of kinetic and structural data currently prevents this validation. [18]…”

Das Spliceosom als Angriffspunkt für Pharmaka

“…[18] Tr otz des Fehlen von Strukturdaten wurden detaillierte SARs der drei Hauptfamilien von SPLMs bestimmt. [18] Tr otz des Fehlen von Strukturdaten wurden detaillierte SARs der drei Hauptfamilien von SPLMs bestimmt.…”

“…[18] Mithilfe von Protein-Protein-Vernetzungen konnten sie nicht nur die Struktur der HEAT-Superhelix lçsen, sondern auch seine Kontakte mit SF3b130, SF3b10 und SF3b14B und die Nachbarschaft zu p14 und U2AF6 nachweisen. [18] Mithilfe von Protein-Protein-Vernetzungen konnten sie nicht nur die Struktur der HEAT-Superhelix lçsen, sondern auch seine Kontakte mit SF3b130, SF3b10 und SF3b14B und die Nachbarschaft zu p14 und U2AF6 nachweisen.…”

“…Bislang gibt es nur wenig Strukturinformationen über die Bindestelle von SPLMs an SF3b;lediglich Strukturvergleiche aus Mutationsstudien geben Hinweise auf eine verläufige Bindungsregion. [18] Tr otz des Fehlen von Strukturdaten wurden detaillierte SARs der drei Hauptfamilien von SPLMs bestimmt. [9] Vonbesonderem Interesse ist eine Arbeit, in der vier Isomere von FD-895 synthetisiert wurden.…”