Fig. 3: RNA recognition by PHAX.
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, The sequence alignment of PHAX proteins covering the cap- and CRM1-binding regions. Identical residues are in brown boxes. Blue squares indicate residues involved in the interaction with cap, yellow squares show residues interacting with CRM1 and gray squares show residues binding RNA. c, Ribbon representation of the RNA recognition by the snRNA export complex. d, Details of the interactions of PHAX E150 with key CBP20 cap-binding residues. e, Details of the specific recognition of the first-transcribed residue by PHAX. f, Summary of the ITC measurements of binding affinities (expressed as the dissociation constant, Kd) between CBC, PHAX103–196 and the cap structure. g, Overlay of Superdex 200 gel filtration elution profiles of the snRNA complex reconstitutions using either WT or Y154A mutant of MBP-PHAX103–196 and a short capped 14-nt RNA. The WT complex elutes in fractions highlighted in red. h, SDS-PAGE analysis of fractions 1–12 of the gel filtration elution profiles shown in g. MBP-PHAX103–196 Y154A is unable to form the complex with CBC and CRM1–RanGTP. The fractions corresponding to the WT complex elution peak are highlighted with the red rectangle. L, input sample loaded onto the column; M, Mw marker. i, Western blotting analysis of streptavidin pulldowns from lysates of HA-dTAG-PHAX mES cells stably expressing MYC-mTurbo tagged PHAXWT, PHAXR137A or PHAXY144E as described in Fig. 2j. j, A schematic model of snRNA export formation based on RNA- and CRM1–RanGTP-triggered folding of PHAX. k, Details of direct interactions between CRM1 and the PHAX region downstream NES.
