Validation and classification of RNA binding proteins identified by mRNA interactome capture

EB1 interaction with RNA involves its MT binding surface. (A) A schematic of EB1 domain organization: EB1 comprises a calponin homology domain (CH) and an EB1-homology (EBH) coiled-coil domain connected by a linker region; (B) EB1 binds RNA in vitro: EMSA of recombinant dmEB1 (left panel) with ³²P-labeled poly(U) 25-mer (lower panel). EB1 (0,5; 1; 5; 10; 20; 40 mM protein) was mixed with 2.5 nM of ³²P-labeled poly(U) 25-mer. The first lane is probe alone control; (C) CSPs along the EB1 N + linker region upon titration with poly(U) 25-mer; (D) CSPs are plotted on the homology model of the dmEB1N domain in ribbon (left panel) and surface (right panel) representations. (E) Interaction of EB1 with RNA maps to the same surface with which EB1 interacts with MTs: hsEB3 bound to microtubules based on 3JAL (Zhang et al. 2015b); (F) RNA competes with MTs for binding to EB1: Cosedimentation of EB1 with MTs and RNA. EB1 alone or preincubated with increasing concentrations of poly(U) 25-mer were added to polymerized microtubules (left panel). After incubation, ultracentrifugation over a sucrose cushion was performed, followed by separation of the pellet and supernatant fractions. The proteins and RNA were separated on SDS PAGE and on urea denaturing gel, respectively (right panel).