Dissecting FRGY: Characterization of Specific FRGY2A/B Isoforms in Relation to Protein-Protein Interactions and Translational Masking.

Abstract

The aim of this project is to investigate the binding properties and functions of three isoforms of the mRNA-binding protein FRGY2: FRGY2A1, FRGY2A2, and FRGY2B (referred to collectively here as FRGY2A/B). Previous research has mainly addressed FRGY2A/B as one large group, but there may be important differences between the known isoforms. Constructs were designed to express His-tagged FRGY2A/B isoforms in E. coli for Ni2+ column purification by FPLC, and constructs designed for expression in in vitro transcription and translation systems. Spectrophotometric data from the purification process, and additional crosslinking provides compelling evidence that FRGY2A/B isoforms self-interact and form multimeric complexes through weak interactions. Further co-immunoprecipitation suggests that isoforms, particularly FRGY2A1 and FRGY2A2 interact with one another, irrespective of phosphatase or kinase treatment. Functional experiments show transcriptional masking in in vitro transcription and translation assays. In this setting, masking appears enhanced by dephosphorylation by calf intestinal phosphatase.