Bcl-2-associated transcription factor 1 interacts with fragile X-related protein 1

Abstract

The absence of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), which is the leading cause of hereditary mental retardation. Fragile X-related protein 1 (FXR1P), which plays an important role in normal muscle development, is one of the two autosomal paralogs of FMRP. To understand the functions of FXR1P, we screened FXR1P-interacting proteins by using a yeast two-hybrid system. The fragile X-related gene 1 (FXR1) was fused to pGBKT7 and then used as the bait to screen the human fetal brain cDNA library. The screening results revealed 10 FXR1P-interacting proteins including Bcl-2-associated transcription factor 1 (BTF). The interaction between FXR1P and BTF was confirmed by using both β-galactosidase assay and growth test in selective media. Co-immunoprecipitation assay in mammalian cells was also carried out to confirm the FXR1P/BTF interaction. Moreover, we confirmed that BTF co-localized with FXR1P in the cytoplasm around the nucleus in rat vascular smooth muscle cells by using confocal fluorescence microscopy. These results provide clues to elucidate the relationship between FXR1P and FXS.

Keywords: Bcl-2-associated transcription factor 1; fragile X-related protein 1; protein interaction.

Figure 1.

Screening FXR1P-interacting proteins by the yeast two-hybrid system (A) After mating AH109-pGBKT7-FXR1 with Y187-pGADT7-cDNA, we screened with SD lacking histidine, tryptophan, and leucine (SD/Trp⁻/His⁻/Leu⁻) medium and obtained 156 positive colonies. (B) After the colonies appeared on the plates, β-galactosidase assays were performed and we obtained 10 different colonies.

Figure 2.

Sequencing result of the BTF positive colonies The 10 positive colonies were sequenced.

Figure 3.

Co-immunoprecipitation of Myc-BTF and HA-FXR1P FXR1P interacts with BTF in vitro. Rat VSMCs were co-transfected with pCMV-Myc-BTF and pCMV-HA-FXR1. Then, the lysates were incubated with a mouse anti-HA antibody (or a mouse anti-Myc antibody) and the protein A/G-Sepharose beads. The complex was segregate by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-Myc (or anti-HA) antibody. The expressions of BTF and FXR1P were detected with anti-Myc and anti-HA antibody, respectively.

Figure 4.

Subcellular co-localization studies of FXR1P and BTF (A) Left: rat VSMCs were transfected with pEGFP-N1-FXR1. FXR1P (green) is located in the cytoplasm. (B) Left: rat VSMCs were transfected with pDsRed-Monomer-N1-BTF. BTF (red) is located in the cytoplasm. The nuclei were stained by DAPI (middle; panels A and B). All of the samples were visualized by laser confocal microscope 48 h after transfection.

Figure 5.

Co-localization studies of FXR1P and BTF pEGFP-N1-FXR1 and pDsRed-Monomer-N1-BTF were co-transfected together into the rat VSMCs. All of the samples were tested after transfection for about 48 h. FXR1P (green) and BTF (red) are visible around the nucleus. Positive signal (yellow) is visible in the cytoplasm. Yellow indicated the co-localization of pEGFP-FXR1P with pDsRed-BTF. The nuclei (blue) were stained by DAPI.