Screening Molecules Interacting With MINT In Yeast Two-hybrid System

Mint, a protein with 3576 amino acids residues, is rich with Pro and Ser and is located in nuclear matrix. Protein sequence analysis reveals that MINT has 3 N-terminal RNA recognition motifs (RRMs) and 4 nuclear localization signals (NLS), so it is sorted to spen family. It can competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. MINT null mutant mice were embryonic lethal, with multiple organs constructal abnormal. This suggests MINT plays an improtant role in development. But the exact mechanism how it functions is not very clear.To study the mechanism of the repression, yeast two-hybrid assay was performed to screen proteins that can interact with MINT. Because of its unusual length(3576 amino acids), MINT is divided into 6 fragments to do the research. Here we report the results of screening with MINT-F1 (amino acids residues 1-365),MINT-F3 ( amino acids residues 1015-1661 ) and MINT-F4(amino acids residues 1662-2228). The bait vector is constructed by inserting the gene fragments into pGBKT? to screen the cDNA library of human lymph nodes or mouse embryo E9.Sequening of the positive clones show that there .are 2 significative molecules interacting with MINT-F1: Vav oncogene and microspherule protein l(MCRSl). Vav is a member of the dbl family of guanine nucleotide exchange factors (GEF) for the rho/rac family of GTP binding proteins which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several signal transduction domains which can link cell surface receptors to downstream signaling proteins, and might play an important role in hematopoiesis. MCRS1 can interact with pi20, a proliferation-related nucleolar protein. MCRS1 may also affect the size and shape of the nucleolus. From the results, we can see that the function of Mint may be complicating, it may participate in several signal pathways, or affect the cell cycle and the function of nucleolus.No positive clone was screened with MINT-F3 or MINT-F4. It indicates that the protein MINT might be shaped as a dumbbell, the functional domains are located at the N- and C-terminals, with a frame area connecting them.