To produce mRNA, the spliceosome excises introns from pre-mRNAs in two sequential reactions – branching and exon ligation. During this process of splicing, the spliceosome recognizes the 5’ and 3’ ends of the intron as well as a conserved adenosine nucleotide (termed branch site) and undergoes a dynamic rearrangement between these two reactions. This rearrangement is mediated by Prp16, as protein that uses energy from breaking down ATP to remodel the spliceosome after branching.
In this work we used budding yeast as a model organism to purify native spliceosomes that were trapped right after branching by using a mutation in Prp16 that blocks remodeling after the first catalytic step. We then prepared a frozen sample of these spliceosomes and utilized an electron microscope operated under cryogenic conditions (cryo-EM) to visualize individual spliceosome particles and obtain a 3D image of the complex that allowed us to build a near-atomic model of all of the spliceosome’s RNA and protein components. The resulting structure of the spliceosome in the branching conformation confirmed previous biochemical studies and showed that the active site is composed of RNA, whereas proteins promote formation for he active site and docking of the pre-mRNA substrate in the proper configuration necessary for catalysis. Importantly, the structure elucidated the molecular basis for recognition of the 5' end of the intron and of the branch adenosine.
To elucidate the structural consequences of Prp16 action, we then assembled spliceosomes on a pre-mRNA substrate containing a mutation that allows the Prp16 remodelling step but prevents mRNA formation. We then used cryo-EM to obtain a 3D image and build a near-atomic model of the spliceosome right before exon ligation. We found that the main consequence of Prp16 activity is to undock the RNA helix containing the branch site from the catalytic core in order to allow docking of the 3’ end of the intron. When undocked from the active site, the branch helix in the second step conformation would clash with first step proteins, thus explaining why dissociation of first step proteins is required for the second step of splicing. Moreover, our structure revealed how second step proteins stabilize the undocked conformation of the branch helix and suggests a plausible model for how the 3’ end of the intron binds in the active site.
Our two spliceosome structures in the branching and exon ligation states reveal the two active conformations of the spliceosome and elucidate the molecular basis for dynamics of the catalytic spliceosome.
