Fig. 2: PHAX-mediated contacts between CBC and CRM1.
From: Structural basis for the synergistic assembly of the snRNA export complex
a, Schematic representation of the PHAX domain structure. ARM, ARS2 recognition motif; ST2, phosphorylation site cluster 2; NES, nuclear export signal; RBD, RNA binding domain. b, Sequence alignment of PHAX proteins covering the CBC-binding and NES regions. Identical residues are in brown boxes. Blue squares indicate residues involved in the interaction with CBC, and yellow squares show residues interacting with CRM1. c, Ribbon representation of the CBC–CRM1 interactions mediated by PHAX and CBP20. The three black rectangles position the close-up views shown in d–f. d, Details of the interactions between the PHAX helix (residues 117–136, dark brick) and CRM1 shown as surface (yellow). The NES residues of PHAX insert into hydrophobic pockets on CRM1. Several charged contacts with CRM1 are shown. e, Details of the hydrophobic and charged interactions between the PHAX helix (residues 117–136, dark brick) and CBP80 shown as surface (light blue). f, Molecular details of the interactions between the CRM1 (yellow) and CBP20 (dark blue). g, Overlay of Superdex 200 gel filtration elution profiles of the snRNA complex reconstitutions using either WT or W118E mutant of MBP-PHAX103–196 and a short capped 14-nt RNA. The WT complex elutes in fractions highlighted in red. h,i, SDS-PAGE analysis of fractions 1–12 of the gel filtration elution profiles of WT (h) and W118E mutant (i) of MBP-PHAX103–196 shown in g. The red rectangle shows the fractions containing the snRNA export complex. In i, MBP-PHAX103–196 W118E is unable to form the complex with CBC and CRM1–RanGTP. L, input sample loaded onto the column; M, Mw marker. j, Western blotting analysis of streptavidin pulldowns from lysates of HA-dTAG-PHAX mES cells stably expressing MYC-mTurbo tagged PHAXWT or PHAXW108A with the parental cell line (−) serving as a negative control. Cells were treated with dTAGV-1 for 4 h to deplete endogenous HA-dTAG-PHAX protein and treated with biotin for a further 4 h to induce PL. Input (left) and streptavidin-enriched pulldown (PD; right) samples were probed with antibodies against MYC, CBP20, CBP80, Ran, CRM1 and ARS2 with ACTIN serving as an input loading control. k, A schematic model of the impact of hW118A mutation on the snRNA export complex assembly.
