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Identification and characterization of small molecule inhibitors of pre-mRNA splicing that block spliceosome assembly at novel stages

dc.contributor.advisorLührmann, Reinhard Prof. Dr.
dc.contributor.authorSidarovich, Anzhalika
dc.titleIdentification and characterization of small molecule inhibitors of pre-mRNA splicing that block spliceosome assembly at novel stagesde
dc.contributor.refereeStark, Holger Prof. Dr.
dc.description.abstractengThe spliceosome is a highly dynamic, megadalton ribonucleoprotein (RNP) molecular machine that catalyses the important step of the post-transcriptional processing of eukaryotic precursor messenger RNA (pre-mRNA), called splicing. During splicing the non-coding nucleotide intervening regions (introns) of newly transcribed pre-mRNA are excised and the protein-coding sequences (exons) are ligated via two consecutive transesterification reactions. The spliceosome is assembled de novo on each pre-mRNA intron by consecutive recruitment of five RNA-protein complexes (snRNPs) and numerous non-snRNP factors in a dynamic manner. Initially, the U1 and U2 snRNPs bind to the 5’ splice site and branch point sequence, respectively, forming the A complex. In the next step, the preformed U4/U6.U5 tri-snRNP binds and complex B is assembled. Thereafter, complex B undergoes multiple rearrangements in terms of both its conformation and composition, including the dissociation of U1 and U4, resulting in the formation of the Bact complex. After the formation of the catalytically active B* complex, the first step of splicing takes place, generating the cleaved 5' exon and intron-3' exon lariat intermediates, and creating complex C, which performs the second step of splicing, resulting in the ligation of the 5' and 3' exons and release of an intron in the form of a lariat. Various assembly intermediates of the spliceosome have been experimentally defined and characterized. However, the dynamics of protein exchange, as well as RNA rearrangements during the spliceosome assembly remain in many aspects unclear. The spliceosome assembly is highly regulated in multiple ways. Disruption or misregulation of alternative and constitutive splicing are the cause or severity modulator of many human diseases, including among others cancer, neurodegenerative and autoimmune diseases, making the spliceosome a highly attractive drug target. Small molecule inhibitors that block discrete steps of the extremely dynamic assembly and functional cycle of the spliceosome are not only of potential therapeutic value, but also highly useful for the detailed investigation of the structure and function of the spliceosome. However, only a limited number of small molecule inhibitors that specifically target the pre-mRNA splicing machinery have been identified at present. Using a robust, rapid and sensitive high throughput in vitro splicing assay, which monitors the formation of step I spliceosomes (i.e. the spliceosomal C complex) by measuring the association of a FLAG-tagged version of the DEAD box ATPase Abstrakt, a chemical library of ~172,000 small molecules was screened for splicing inhibition activity. All compounds showing at least a 50% decrease in the signal intensity of bound Abstrakt were subjected to a second test, and reproducible hits were finally tested in an in vitro splicing assay using 32P-labeled adenovirus-derived MINX-MS2 pre-mRNA as substrate. Ten compounds were confirmed to be inhibitors of pre-mRNA splicing in vitro, and exhibited IC50 values ranging from 3 to 50 µM, To determine at which stage they inhibit splicing, I performed a splicing time course and analysed the spliceosomal complexes formed by native agarose gel electrophoresis. Analysis of splicing complex formation revealed that at least one compound (hereafter designated as 028), led to an accumulation of A complexes and a complex denoted as B028 that sediments slightly faster than the pre-catalytic B complex on glycerol gradients. Gradient centrifugation experiments indicated that U4/U6.U5 tri-snRNP stability is not affected significantly by 028. To characterize in more detail spliceosomal complexes formed in the presence of compound 028, I subjected the stalled splicing reactions to glycerol-gradient centrifugation and purified the complexes in a given peak by MS2-MBP affinity selection. Initial analysis of the RNA and protein composition of complexes affinity- purified from the “B-like” peak, suggested that compound 028 stalls splicing at a novel stage of the spliceosome activation step; i.e., inhibition appears to take place after release of the U4 snRNP, but prior to the release of the LSm and B-specific proteins and before the stable integration of the Prp19 complex, the recruitment of Bact specific proteins and the phosphorylation of U2 protein SF3b155. Analysis of the RNA-RNA network within B028 by RNA structure probing and psoralen crosslinking suggested, but did not definitively prove, that the catalytically important U6-ISL and U2/U6 helix Ia and Ib likely are formed in the spliceosomes stalled by compound 028. Purified B028-complexes could be chased into catalytically active spliceosomes upon addition of microccocal nuclease-treated nuclear extract, demonstrating that they are functional complexes. We thus performed initial “chase” experiments with purified B028 complexes to follow the recruitment and release of proteins during the activation step. Our results suggest that ATP is required for the recruitment of the Prp19/CDC5L complex proteins and B-specific proteins. Negative stain electron microscopy of B028-complexes and 3D reconstruction of the B028-particle revealed a unique morphology, which is different from the pre-catalytic B and activated Bact spliceosomal complexes, but suggest common morphological features with both B and Bact, consistent with them being stalled at an intermediate assembly stage. Finally, structure-activity relationship (SAR) studies, in which modifications of compound 028 were assayed for their effect on pre-mRNA splicing in vitro, revealed structural determinants that are important for the inhibition activity of compound 028. Thus, compound 028 allows us to obtain a novel snapshot of the spliceosome assembly pathway and to perform detailed structural and functional investigations, to improve our limited understanding of the dynamic rearrangement of spliceosomal components during spliceosome
dc.contributor.coRefereeKrebber, Heike Prof. Dr.
dc.subject.engSplicing; spliceosomede
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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