| Summary: | 碩士 === 國立陽明大學 === 解剖暨細胞生物學研究所 === 92 === Alternative splicing of pre-mRNA is a regulatory mechanism of gene expression in eukaryotic cells. This pathway can generate different mRNA isoforms, and thereby increases the coding capacity of genes. SR (Ser/Arg dipeptide-rich) proteins and hnRNP proteins play important roles in constitutive and/or regulated splicing. RBM4 is a non-SR splicing regulatory protein, and shares a same import pathway with SR proteins to the nucleus. RBM4 possesses two RNA recognition motifs (RRM) and an alanine-rich C-terminal domain (CAD). Both RRM and CAD are critical for alternative splicing. We postulate that the CAD of RBM4 acts as a module for protein-protein interaction. In this study, we employed a yeast two-hybrid system to search for proteins that interact with the CAD. The screens yielded five positive clones. The candidate protein hnRNP M belongs to the subfamily of hnRNPs that participate in various steps of RNA metabolism, including pre-mRNA splicing. The interaction of RBM4 with hnRNP M was confirmed by in vitro binding assays. We demonstrated that hnRNP M binds to RBM4 through its methionine/argenine-rich region (M/R-rich motif). Transient expression of hnRNP M in HEK293 cells can differentially affect alternative splice site or exon selection. More interestingly, the M/R-rich motif can replace the CAD of RBM4 in modulation in alternative splicing.
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