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Biochemical and structural characterization of spliceosomes purified at defined stages of assembly from the yeast S. cerevisiae

dc.contributor.advisorLührmann, Reinhard Prof. Dr.
dc.contributor.authorDannenberg, Julia
dc.titleBiochemical and structural characterization of spliceosomes purified at defined stages of assembly from the yeast S. cerevisiaede
dc.contributor.refereeLührmann, Reinhard Prof. Dr.
dc.description.abstractengSplicing of pre messenger RNA (pre mRNA) is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) comprising several small nuclear (sn) RNPs and numerous proteins. The spliceosome assembles on its pre mRNA substrate in an ordered process that begins with recognition of the 5' end of the intron (5' splice site, 5'SS) by the U1 snRNP. Thereafter the U2 snRNP binds to the pre mRNA's branch point sequence (BPS), forming complex A. Complex A then binds the pre-formed U4/U6•U5 tri snRNP to form complex B, which contains a full set of snRNAs in a pre-catalytic state. Complex B is then activated for catalysis by a major rearrangement of its RNA network and its overall structure; this remodeling includes dissociation of the U1 and U4 snRNAs and the formation of the activated spliceosome Bact. In the catalytically activated complex (termed B*) step 1 catalysis takes place: the adenosine at the BPS attacks the 5'SS, generating a cleaved 5' exon and intron 3' exon intermediate. The resulting complex C then catalyzes step 2 catalysis, in which the intron is cleaved at the 3' splice site (3'SS) with concomitant ligation of the 5' and 3' exons to form mature mRNA. Thus, the spliceosome is a particularly dynamic RNP machine that undergoes many changes in composition and conformation. The structural dynamics of the spliceosome are facilitated by the action of multiple DExD/H box RNA helicases. Among these are Prp5 and Prp2, which are essential ATPase required prior to the first step of pre-mRNA splicing. Prp5 enables stable U2 snRNP association with the branch site and Prp2 promotes a structural rearrangement that transforms the Bact into the catalytically activated B* complex.  The pathway of spliceosome assembly and the main features of its catalytic chemistry appear to be conserved between metazoans and yeast. Thus, I set out to study isolated spliceosomal complexes from the lower eukaryote Saccharomyces cerevisiae as it was already known to possess a basic (constitutive) spliceosomal machinery similar to that of primates. Each new round of splicing generates a catalytic centre de novo during the transitions from complex B to Bact to B* and to the product of step 1 of splicing, complex C. Here, I focused on the isolation and characterization of complex B, the activated complex Bact and complex C. Each complex was stalled via truncation or modification of the actin pre-mRNA used during the splicing reaction or adjustment of the ATP concentration in the splicing reaction. Each complex was then isolated by centrifugation and affinity selection, their proteomes determined by mass spectrometry (in collaboration with Prof. Henning Urlaub) and their structures examined by electron microscopy (in collaboration with Dr. Berthold Kastner and Prof. Holger Stark), for the first time. The analysis of the three stalled yeast spliceosomal “snapshots” isolated here has made it possible to pinpoint the window of function for important spliceosomal proteins. For instance, the number, characteristics and time of recruitment of evolutionarily conserved proteins involved in the formation and stabilization of the U2/U6/pre-mRNA network of the catalytic center in complex Bact remained so far elusive. The data revealed several evolutionarily conserved proteins recruited at the time of pre-catalytic activation (i.e. Cwc2, Cwc24, Yju2, Prp2 and Spp2) and also provided important hints for those proteins involved in promoting step 1 catalysis and the formation of complex C (i.e. Cwc25). Compared to metazoan spliceosomes the number of proteins associated with purified yeast spliceosomes at any stage is less than a half, yet more than 85% of these have evolutionarily conserved counterpart in humans. The less complex protein composition of yeast spliceosomes offers also a significant advantage for three dimensional (3D) structure analyses. The EM analyses show for the first time images of S. cerevisiae spliceosomal complexes at well defined stages of function. These are at an unprecedented quality level, and they are well suited for 3D structure investigations, based on criteria such as their structural integrity and homogeneity.  In this work I have also applied dual-color fluorescence cross-correlation spectroscopy (dcFCCS, in collaboration with Prof. Jörg Enderlein, III. Institute of Physics, University of Göttingen), to measure the binding affinity of splicing factors – which were detected in the first part of this work – to the yeast spliceosome and to follow their binding dynamics during the catalytic activation of the spliceosome promoted by the RNA helicase/ATPase Prp2 and its co-activator Spp2. dcFCCS is a sensitive and versatile optical technique that allows the direct analysis of the dynamic association and dissociation events among proteins and/or RNAs in complex systems in solution at low nanomolar concentrations and in equilibrium, without requiring biochemical or physical perturbation of the sample. For this purpose I have employed a recently described purified splicing system developed in our laboratory which recapitulate catalytic activation and step 1 of splicing in vitro and consists of Bact complexes assembled in extracts where Prp2 is thermo sensitive and can be heat-inactivated. These Bact complexes lacking Prp2 are then purified to near homogeneity and complemented with recombinantly expressed Prp2 and Spp2 splicing factors. To perform dcFCCS, the Bact complexes were assembled on pre mRNA labeled with a red fluorescent dye. In addition, spliceosomal proteins of interest were labeled in vivo by fusing them with the green fluorescent protein EGFP, by genetic modification in yeast. The purified doubly-labelled spliceosomes were then analyzed before and after catalytic activation by Prp2 and the weakening or strengthening of the binding of the EGFP-labeled protein to the spliceosome was analyzed by measuring the cross-correlation between the green label and the red fluorescent dye. The analysis and evaluation of the dcFCCS data in this work was done in collaboration with Prof. Jörg Enderlein and Mira Prior (III. Institute of Physics, University of Göttingen).  The data revealed that the binding affinity of quite a number of proteins is significantly changed during the Prp2-mediated catalytic activation of the spliceosome. Specifically, the essential zinc finger protein Cwc24, was quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. Interestingly, the U2-associated SF3a and SF3b proteins Prp11 and Cus1 were destabilized during catalytic activation. Indeed, they remained bound to the B* spliceosome under near-physiological conditions (i.e. 75 mM), but their binding was reduced at higher salt. As the U2 SF3a/b proteins bind near the branch point sequence, this indicated that the branch site must be remodeled in complex B* as a prerequisite for step 1 catalysis. On the other hand, high affinity binding sites were created for the step 1 factors Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. In conclusion, These results shed light on the nature of the structural remodeling mediated by Prp2 in the spliceosome and suggest that during catalytic activation the spliceosome undergoes significant rearrangements. de
dc.contributor.coRefereeFicner, Ralf Prof. Dr.
dc.subject.engpre-mRNA splicingde
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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