| dc.contributor.advisor | Stark, Holger Prof. Dr. | |
| dc.contributor.author | Townsend, Cole | |
| dc.date.accessioned | 2022-01-27T12:34:00Z | |
| dc.date.available | 2022-02-03T00:50:08Z | |
| dc.date.issued | 2022-01-27 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0008-5A07-C | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-9051 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Structural characterization of human spliceosome activation by cryo-EM | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Stark, Holger Prof. Dr. | |
| dc.date.examination | 2021-12-08 | |
| dc.description.abstracteng | 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 (B<sup>act</sup> 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 (Mg<sup>2+</sup>) 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-B<sup>act-1</sup> and pre-B<sup>act-2</sup>. The pre-B<sup>act</sup> 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. | de |
| dc.contributor.coReferee | Urlaub, Henning Prof. Dr. | |
| dc.contributor.thirdReferee | Stein, Alexander Dr. | |
| dc.contributor.thirdReferee | Lührmann, Reinhard Prof. Dr. | |
| dc.contributor.thirdReferee | Tittmann, Kai Prof. Dr. | |
| dc.contributor.thirdReferee | Faesen, Alexis Caspar Dr. | |
| dc.subject.eng | cryo-EM, RNA, spliceosome, splicing | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-5A07-C-1 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2022-02-03 | |
| dc.identifier.ppn | 1787356035 | |