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Investigation of the structure of spliceosomal complexes from the yeast S. cerevisiae

dc.contributor.advisorLührmann, Reinhard Prof. Dr.
dc.contributor.authorKumar, Vinay
dc.titleInvestigation of the structure of spliceosomal complexes from the yeast S. cerevisiaede
dc.contributor.refereeLührmann, Reinhard Prof. Dr.
dc.description.abstractengNuclear pre-mRNA splicing is catalysed by the spliceosome, a multi-megadalton ribonucleoprotein (RNP) complex. It assembles de novo for each round of splicing on premRNA intron with stepwise binding of five small nuclear ribonucleoprotein particles (snRNPs), and numerous proteins. The process of splicing initiates with the U1 and U2 snRNPs associating with the pre-mRNA’s 5’ splice site (SS) and branch site (BS), respectively, giving rise to the A complex. The newly formed A complex is further joined by the U4/U6.U5 tri-snRNP, which results in the formation of the precatalytic B complex. During the highly choreographed event of spliceosomal activation, Brr2 dissociates U4 RNA from U6 RNA, which are base-paired together in the tri-snRNP, and U6 RNA restructures and together with U2 RNA forms the active site. It has always been a major question, how Brr2 is prevented in isolated U4/U6.U5 tri-snRNPs from pre-maturely dissociating the U4/U6 RNAs. In the human tri-snRNP Brr2 is situated away (~10 nm) from its RNA substrate, the U4/U6 duplex. The human Sad1 protein has a key role in stabilization the particle. Sad1 is present in stoichiometric amounts and it is located in a strategically important position at the interface between U4/U6 and U5 snRNP where it maintains numerous protein-protein contacts to act as a stabilizing clamp between the two snRNPs and at the same time inhibits premature access of Brr2 to the U4 RNA. In yeast instead, the reported tri-snRNP structures show that Brr2 is already loaded onto its U4 snRNA substrate, and ready to unwind the U4/U6 duplex. In fact, in the presence of ATP it does unwind and dissociate the tri-snRNP while the human tri-snRNP remains stable. The stability difference in the presence of ATP has been attributed to the presence of Sad1 in the human tri-snRNP, while Sad1 is usually not observed in isolated yeast tri-snRNPs. In order to prove the proposed role of Sad1 I set out to isolate and characterize yeast tri-snRNP particles which are similar to human tri-snRNPs in terms of protein composition and biochemical behaviour. The challenge rest in finding biochemical conditions and modified purification protocols, which would provide such yeast tri-snRNPs, which are then used for biochemical and structural comparison with the human tri-snRNP. Yeast tri-snRNPs were purified using a two-step procedure, using a Sad1- TAP tag and gradient centrifugation under suitable buffer conditions. I succeeded in purifying a tri-snRNP which is stable under ATP conditions. This tri-snRNP for the first time contained a stably associated Sad1 and Prp28. It appeared that both proteins may have an important role in stabilizing the yeast tri-snRNP. Additionally, I found that Sad1 and Prp38 are mutually exclusive proteins. Despite the drastically changed biochemical characteristics of the tri-snRNP purified under the novel conditions, Brr2 still appears to adopt the typical yeast-like
dc.contributor.coRefereeFicner, Ralf Prof. Dr.
dc.contributor.thirdRefereeUrlaub, Henning Prof. Dr.
dc.contributor.thirdRefereeStark, Holger Prof. Dr.
dc.contributor.thirdRefereeGroßhans, Jörg Prof. Dr.
dc.contributor.thirdRefereeKehlenbach, Ralph Prof. Dr.
dc.subject.engSplicing + Tri-snRNP + Sad1de
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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