Single-Molecule Fluorescence Spectroscopy of the Structure and Dynamics of the Spliceosomal Complex

Abstract

Binding reactions can be ideally investigated by Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS). Here, this method is used to study the recruitment and dissociation of proteins to and from the spliceosome. The spliceosome is the cellular machinery responsible for removing non-coding introns from precursor mRNA (pre-mRNA). The spliceosome assembles on a pre-mRNA, which consists of two exons and one intron, stepwise by the binding of five snRNPs and several other proteins. This leads to the formation of the B complex which has no active catalytic center yet. Structural rearrangements lead to the activated Bact complex which is formed by further rearrangements in the catalytically active B* complex. The B* complex catalyzes the first splicing step in which the branch site (BS) attacks the 5' splice site (5'SS). The 5' exon is cleaved and the intron-3'-exon lariat is formed. The resulting C complex then catalyzes the second splicing step. The intron is cleaved and both exons are joined together forming mature mRNA. During both catalytic steps, the spliceosome undergoes many changes in its protein composition, and several structural rearrangements occur. These changes and rearrangements can be investigated by standard biochemical methods. However, these methods often do not provide data about dynamics, which are necessary for understanding the recruitment and release of particular molecules. One method which provides these information is FCCS. In contrast to methods like mass spectrometry, in FCCS measurements it is possible to observe reactions in real-time and at equilibrium without further biochemical perturbation of the sample. FCCS works at low nanomolar concentrations and only small sample volumes are necessary. Using FCCS, it could be determined how changes in the spliceosome composition and conformation occur (simultaneously or consecutively). The roles of certain spliceosomal RNA helicases in the restructuring of the complex could be investigated. FCCS enables the observation of protein-protein interactions and the determination of binding constants for proteins to the spliceosome. In order to better understand the dynamic nature of the spliceosome during its catalytic activation, the step 1 factor Cwc25 and the step 2 factors Slu7, Prp18, and Prp16 were investigated. Their role in the maturation process, the fundamental question of the time point and manner of their recruitment has not been answered yet. With FCCS, the binding of the step 1 factor Cwc25 was observed. It was shown that Cwc25 has a high-binding affinity to the spliceosome after the Prp2-mediated rearrangements into the catalytically activated B* complex. The high-binding affinity was reflected in a strong binding constant of 30 pM which was measured with FCCS. By using several mutant pre-mRNAs it could be demonstrated that Cwc25's release does not depend on the second catalytic step per se. Its dissociation depends on the docking of the 3'SS to the active site and the action of Prp16 and Slu7/Prp18. It was also observed that the distance between the branch site and 3'SS influences the release of Cwc25 and the second catalytic step. If the distance between BS and 3'SS is short (e.g. 7 nucleotides long), the distance between the 3'SS and the active site is also small. The 3'SS can dock into the active site without the further stabilization by Slu7/Prp18. The activity of Prp16 and the docking event lead to the release of Cwc25. Slu7/Prp18 can stabilize the system and thereby induce further release of Cwc25. If the distance between BS and 3'SS is longer (e.g. 38 nucleotides), Slu7/Prp18 has to stabilize the interaction of the 3'SS and the active site and the step 2 conformation. In this case, Prp16 alone cannot induce the release of Cwc25. Cwc25 does dissociate from the spliceosome after the action of Prp16 and in the presence of Slu7/Prp18. It was shown further that Prp16 and its ATPase activity are necessary for the formation of a functional step 2 active site. Slu7/Prp18 are required for the efficient docking of the 3' splice site (3'SS) to the active site. The FCCS experiments showed that Prp16 and Slu7/Prp18 have distinct binding sites in the spliceosome which are formed during the catalytic activation. During activation their first low-affinity binding sites are transformed into high-affinity binding sites. Both proteins are bound to the spliceosome at an early stage so that they are present before their catalytic function is actually required in the process. Here, it was shown that FCCS is ideally suited to investigate macromolecular protein complexes like the spliceosome and is a powerful tool for studying quantitatively spliceosomal protein dynamics at equilibrium.