FIG. 2.

Mapping the domain on dU2AF⁵⁰ that is necessary and sufficient for interaction with dU2AF³⁸. (A) An SDS–12% polyacrylamide gel of dU2AF⁵⁰ wild-type and deletion mutant proteins stained with Coomassie blue. U2AF subunits were coexpressed in E. coli with the dimeric coexpression plasmid. dU2AF⁵⁰ was (his)₆ tagged (His-dU2AF⁵⁰) in the dU2AF heterodimer, and hU2AF³⁵ was (his)₆ tagged in the hU2AF heterodimer (His-hU2AF³⁵). dU2AF heterodimer formation was assessed by copurification of dU2AF³⁸ on Ni²⁺-NTA-agarose. E. coli lysates from uninduced (−) and induced (+) cells as well as the eluate (EL) from Ni²⁺-NTA-agarose purification are shown. The position of dU2AF³⁸ is indicated by an arrow. dU2AF³⁸ runs heterogeneously due to carboxyl-terminal proteolysis. The identity of these polypeptides as dU2AF³⁸ was confirmed by immunoblot analysis (data not shown; Fig. 4A and B). A similar heterogeneity was observed when an amino-terminal (his)₆-tagged dU2AF³⁸ was purified separately (data not shown). The sizes of the protein molecular size markers are indicated in kilodaltons. WT, wild type. (B) Schematic representation of the results of the copurification interaction assay. The (his)₆ tag (His), RS domain (RS), and three RNA recognition motifs (RRM1–3) of dU2AF⁵⁰ are indicated. The different dU2AF⁵⁰ domains are not drawn to scale.