Abstract
The in vivo assembly of uridine-rich small nuclear ribonucleoproteins (U snRNPs), the central catalytic components of the spliceosome, is a highly organised, multi-step process orchestrated by several multi-protein complexes. Structural analyses have provided valuable insights into their overall architecture. However, critical information on the regulation of U snRNP assembly is still lacking. In this study, we used AlphaFold 3 to model the human 6S intermediate complex consisting of full-length pICln and five Sm proteins SmD1/D2/E/F/G. The available crystal structure used truncated non-vertebrate proteins, omitting the highly flexible C-terminal segments to permit crystallisation. However, the C-terminus of pICln has since been recognised as regulatory region, making full-length computational models an appropriate way to elucidate its structural and functional roles. By integrating modelling with biochemical data from previous studies, our results support a model in which the phosphorylation-dependent regulation of the pICln-SmG interface facilitates downstream assembly steps in vertebrates, including the regulated displacement of pICln by the SmD3/B dimer. According to this model, ULK1-dependent serine phosphorylation in the C-terminal α-helix of pICln may abrogate the secondary structure and weakens its interaction with SmG, favouring ring opening. This complementary in silico approach elucidates the roles of regulatory regions in pICln that were previously inaccessible to crystallographic analysis and provides a framework for targeted experimental validation.