Structural characterization of human spliceosome activation by cryo-EM
von Cole Townsend
Datum der mündl. Prüfung:2021-12-08
Erschienen:2022-01-27
Betreuer:Prof. Dr. Holger Stark
Gutachter:Prof. Dr. Holger Stark
Gutachter:Prof. Dr. Henning Urlaub
Gutachter:Dr. Alexander Stein
Gutachter:Prof. Dr. Reinhard Lührmann
Gutachter:Prof. Dr. Kai Tittmann
Gutachter:Dr. Alexis Caspar Faesen
Dateien
Name:eDiss_cryoEM_spliceosome_activation_Townsend_2021.pdf
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Description:Structural characterization of human spliceosome activation by cryo-EM
Zusammenfassung
Englisch
Eukaryotic genes are transcribed as precursor mRNA (pre-mRNA), in which coding regions (exons) are interrupted by non-coding regions (introns). Introns are excised and exons are ligated together in a two-step process termed "splicing" to produce mature mRNA. Both steps of pre-mRNA splicing are catalyzed by RNA within a complex molecular machine consisting of 5 small nuclear ribonucleoproteins (U1, U2, U4/U6.U5 snRNPs) and over 150 proteins: the spliceosome. For each intron to be spliced, the spliceosome is assembled de novo on its pre-mRNA substrate in a stepwise manner catalyzed by DExD/H-box ATPases, which remodel RNA-RNA and RNA-protein interactions of each complex. Spliceosome assembly begins with consecutive association of U1 and U2 snRNPs with the pre-mRNA, followed by the integration of the U4/U6.U5 tri-snRNP and subsequent release of U1 snRNP to form a pre-catalytic spliceosome, or B complex. The B complex lacks a catalytic center, and must therefore be extensively remodeled in a process called activation, to form an activated complex (Bact complex). Spliceosome activation constitutes the largest flux in the composition of the spliceosome, with over 30 proteins being dissociated and more than 25 being integrated to form the activated complex. The activation phase is catalyzed by the DExD/H-box ATPase BRR2, which unwinds the base-pairing between U4/U6 snRNAs, leading to the dissociation of U4 snRNP and numerous proteins, and allowing for the reorganization of U6 snRNA to form intramolecular base-pairing interactions as well as intermolecular base-pairs with U2 snRNA. The resulting U2/U6 RNA-RNA network results in a triple-helix of RNA that coordinates two divalent metal cations (Mg2+) which are involved in splicing catalysis. While structural and biochemical insights have been gleaned about both the pre-catalytic and activated states of the spliceosome, it is unknown whether structurally and compositionally distinct intermediates during the activation phase may exist. Moreover, the role of proteins in facilitating the formation of the RNA-based catalytic center at the core of the spliceosome is unclear. Using a previously identified small molecule chemical inhibitor of pre-mRNA splicing, we isolated spliceosomes stalled at intermediate stages of activation. By employing single particle cryo-EM and image classifications, we identified two novel and distinct states of the spliceosome following the release of U4 snRNP but prior to the formation of an activated complex, which we termed pre-Bact-1 and pre-Bact-2. The pre-Bact structures offer new insights into the massive exchange of proteins during activation as well as the role of these proteins in guiding the formation of the RNA-base catalytic network formed by base-pairing interactions between U2/U6 snRNAs.
Keywords: cryo-EM, RNA, spliceosome, splicing