Probing the Prp40, Snu71, and BBP interaction using GST pull-down assays. (A) GST pull-down assays probing the interaction between Prp40 domains and Snu71. Various GST-fused Prp40 domains were expressed and purified from Escherichia coli (WW including both WW1 and 2, FF1, FF2, FF3, FF4, FF5, FF6, FF4-6) and were used to pull-down Snu71. Pull-down samples after extensive wash were visualized by SDS-PAGE and Coomassie staining. “*” denotes bands corresponding to various GST fusion Prp40 domains (GST-FF2 and FF3 have significant degradation). “+” denotes bands corresponding to Snu71. Red labels indicate Prp40 domains that pulled down Snu71. (B) GST pull-down assays probing the interaction between Snu71 domains and Prp40. Various GST-fused Snu71 domains (residues 97–220, 382–537, 538–620, whereas domains covering other regions of Snu71 cannot be expressed in E. coli) were used to pull down FLAG-tagged Prp40 FF4, FF5, FF6, and FF4-6 domains. All proteins were expressed and purified from E. coli. “*” denotes bands corresponding to GST or GST-fused Snu71 domains. “+” denotes bands corresponding to Prp40 FF4-6 or FF4. Red labels indicate the GST-Snu71 domain (382–537) that stably interacts with Prp40 FF4 and FF4-6. (C) GST pull-down assay probing the interaction between Prp40 domains and BBP. Various GST-fused Prp40 domains as in A were used to pull down BBP. Pull-down samples were visualized on SDS-PAGE with Coomassie stain. “*” denotes bands corresponding to various GST-fusion Prp40 domains (GST-FF2 and FF3 have significant degradation). “+” denotes bands corresponding to BBP. Red labels indicate Prp40 domains that pulled down BBP. (D) A summary of interactions among BBP, Prp40, Snu71, and Luc7 (indicated by a solid line connecting regions of two proteins) as observed in A–C. (E) Key interaction regions mapped on the structures of Snu71, Prp40 (both were predicted by AlphaFold2; Jumper et al. 2021), and Luc7 (determined by cryo-EM; Li et al. 2019). (F) A model of how Luc7, Snu71, and Prp40 interact with each other in the framework of U1 snRNP in the pre-A complex (Zhang et al. 2021).
