The roles of RBM4 in alternative splicing and translational regulation

• Main Authors: Jung-Chun Lin, 林榮俊
• Other Authors: 譚婉玉
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/16080868293987695857

博士 === 臺灣大學 === 分子醫學研究所 === 96 === RNA-binding motif protein 4 (RBM4) is a nucleocytoplasmic shuttling protein and acts as a precursor mRNA splicing regulator. We identified several mRNA targets of RBM4 through immunoprecipitation of RBM4-containing mRNPs followed by the differential display analysis. Among these candidates, α-tropomyosin (α-TM) is known to exhibit skeletal and smooth cell type-specific splicing patterns. We found that the expression level of the skeletal muscle-specific isoform correlated with that of RBM4 in human tissues and also can be modulated by ectopic expression or suppression of RBM4. By using minigene, we demonstrated that RBM4 can activate the selection of skeletal muscle-specific exons via binding to intronic pyrimidine-rich element. Moreover, we found that RBM4 antagonized the effect of another splicing regulator, PTB, in alternative splicing of α-TM. These results demonstrated that RBM4 plays an important role in cell-type specific expression of α-TM. We next provided evidence showing that RBM4 was a phosphoprotein and the phosphorylation level can be enhanced by cell stress, such as arsenite-exposure. By arsenite treatment, RBM4 was phosphorylated at Ser309 and translocated from the nucleus to the cytoplasm and stress granules as well via the MKK3/6-p38 MAPK signaling pathway. We found that RBM4 suppressed the cap-dependent translation in a cis-element-dependent manner. On the other hand, RBM4 could activate internal ribosome entry site (IRES)-mediated translation likely by enhancing the association of translation initiation factor 4A (eIF4A) with IRES-containing mRNAs. Whereas arsenite treatment promoted loading of RBM4 onto IRES -containing genes and enhanced RBM4-eIF4A interaction. Overexpression of RBM4 could mimic the cell stress effect on activation of IRES-mediated translation. Our results proposed a new paradigm for an RNA-binding protein that could act as a suppressor of cap-dependent translation but as an enhancer of IRES-mediated translation in response to cellular stress. We finally demonstrated that RBM4 participated in the microRNA-mediated translational regulation in differentiated myoblast C2C12 cells. At the onset of myogenesis, RBM4 translocated to the cytoplasm and forms certain cytoplasmic foci. Interestingly, several components of miRNP including Ago2 protein and some muscle cell-specific microRNAs, such as miR-1,133 and 206 associated with RBM4. We further observed that RBM4 could coordinately repress the expression of the reporters containing the miR-1-targeting elements. The presence of RBM4 promoted the loading of Ago2 protein onto these reporter mRNAs. Therefore, our results may uncover multiple roles that RBM4 played in different events of RNA metabolism.