Structural and functional studies into branchpoint sequence recognition and the U4/U6 di-Snrnp in cyanidioschyzon merolae

Pre-mRNA splicing ensures mRNA transcripts contain contiguous coding sequences prior to protein synthesis. 5’ splice site recognition of pre-mRNA is unknown in Cyanidioschyzon merolae, lacks the U1 snRNP. Conservation among the core splicing proteins is low, and there is potential for novel adaptations. Similar to yeast, branchpoint sequence recognition appears driven by Msl5 and stabilized by Mud2. Electrophoretic mobility assays showed Msl5, with its canonical KH-QUA2 domain, specifically binds to the conserved branchpoint sequence. Mud2 is a novel ScMud2/U2AF65 homolog. Conversely, U4/U6 di-snRNP assembly appears conserved. Fluorescence polarization assays with the U4 5’ stem loop and the crystal structure of Snu13 revealed a conserved interaction. Snu13 and the Sm heptamer were able to bind base-paired U4/U6. Finally, circular dichroism spectroscopy and in silico mapping of intrinsic disorder found several proteins were partially unstructured; this would allow a minimal spliceosome to functionally fulfill the roles of otherwise necessary proteins.