A trans-acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigene

Two clones were isolated in a three-hybrid screen of a rat fetal brain P5 c DNA library with an intronic splicing enhancer of the amyloid precursor pro tein (APP) gene as RNA bait. These clones represent the rat homologues of t he previously described genes CUG-binding protein (CUG-BP) and Siah-binding protein (Siah-BP). Both interact in a sequence-specific manner with the RN A bait used for library screening as well as with the CUG repeat. In contra st, no interactions were observed in the three-hybrid assay with other bait s tested. In two-hybrid assays, Siah-BP interacts with U2AF65 as well as wi th itself. EWS, an RGG-type RNA-binding protein associated with Ewing sarco ma, was identified as an interacting partner for the CUG-BP homologue in a two-hybrid assay for protein-protein interactions performed with various fa ctors involved in RNA metabolism. Splicing assays performed by RT-PCR from cells cotransfected with certain cDNAs and an APP minigene, used as a repor ter, indicate exclusion of exon 8 if the CUG-BP homologue is present. We co nclude that clone AF169013 and its counterpart in human CUG-BP could be the trans-acting factors that interact with the splicing enhancer downstream o f exon 8, and in this way influence alternative splicing of the APP minigen e.