Fig. 5: ARS2 interaction with the snRNA export complex.
From: Structural basis for the synergistic assembly of the snRNA export complex
a, A schematic representation of the ARS2 and PHAX domain structure. The ARS2 effector domain binds the ARM motif in PHAX N-terminus. ARM, ARS2 recognition motif; ST2, phosphorylation site cluster 2; NES, nuclear export signal; RBD, RNA binding domain. b, A schematic representation of the interaction of the CBC–ARS2147–871–RNA complex with CRM1, RanGTP and PHAX103–196. c, The Superdex 200 gel filtration elution profile of the mixture of the CBC–ARS2147–871–RNA complex with CRM1, RanGTP and PHAX103–196. The CBC–ARS2147–871–RNA complex elutes in fractions highlighted in red. d, SDS-PAGE analysis of fractions 1–12 of the gel filtration elution profile shown in c. The lower panel shows a 7% SDS-PAGE analysis of the same fractions enabling better resolution of CRM1 and ARS2147–871. The red rectangle indicates fractions containing the CBC–ARS2147–871–RNA complex. CRM1, PHAX and RanGTP elute in later fractions. L, input sample loaded onto the column; M, Mw marker. e, A schematic representation of the interaction of the CBC–ARS2147–871–RNA complex with CRM1, RanGTP and FL PHAX. ARS2 binds to PHAX via its ARM motif. f, The Superdex 200 gel filtration elution profile of the mixture of the CBC–ARS2147–871–RNA complex with CRM1, RanGTP and FL PHAX. The snRNA export complex bound to ARS2147–871 elutes in fractions highlighted in red. g, SDS-PAGE analysis of fractions 1–12 of the gel filtration elution profile shown in f. The red rectangle indicates fractions containing the snRNA export complex bound to ARS2147–871. h, A schematic representation of the interaction of the CBC–ARS2147–871–RNA complex with CRM1, RanGTP and E9R mutant of FL PHAX. ARS2 binding to PHAX via its ARM motif is prevented. i, The Superdex 200 gel filtration elution profile of the mixture of the CBC–ARS2147–871–RNA complex with CRM1, RanGTP and FL PHAX E9R. j, SDS-PAGE analysis of fractions 1–12 of the gel filtration elution profile shown in i. The red rectangle indicates fractions where the snRNA export complex bound to ARS2147–871 eluted in g. Here, ARS2 becomes substoichiometric and the snRNA complex elutes later in fractions 4–6. k, Western blotting analysis of streptavidin pulldowns from lysates of HA-dTAG-PHAX mES cells stably expressing MYC-mTurbo tagged PHAXWT and PHAXE9R as described in Fig. 2j. l, A model for the role of ARS2 in the snRNA export complex assembly. ARS2 can simultaneously interact with the ARM motif of PHAX and bind to the groove formed at the CBP20–CBP80 interface. The latter interaction prevents PHAX binding to CBC. In the presence of CRM1–RanGTP and capped RNA, PHAX can displace ARS2 from CBC. ARS2 may remain bound to the complex via the ARM motif but might also be released, enabling its association with other CBC-effectors complexes.
