探討mRNA結合蛋白RBM4於選擇性剪接及轉譯調控之功能

Abstract

訊息RNA (mRNA)藉由替代性剪接造成蛋白質表現的多樣性，此現象在不同組織或發育時期受到嚴密 的調控以符合其生理功能， 多種RNA結合蛋白均具有調控此替代性剪接之功能，RNA binding motif protein 4 (RBM4) 便具有剪接因子之特性。利用免疫沈澱及分示法(Differential display)的方法發現數種可與RBM4形成複合物之mRNA，包括α-tropomyosin (α-TM)。RBM4會促進橫紋肌細胞特有的α-TM表現，此現象與RBM4在橫紋肌細胞有較高的表現量具有其一致性。因此在橫紋肌細胞中表現量較高的RBM4，具有拮抗另一普遍存在於各種細胞中的剪接調控因子polypyrimidine-tract binding protein (PTB)對於橫紋肌細胞特有α-TM表現的抑制作用。此結果證明RBM4在不同細胞的細胞核中具有調控替代性剪接的重要特性。 當細胞處於由砷化物所造成的高氧化狀態下時，原本多位於細胞核之RBM4，會因被大量的磷酸化而累積在細胞質中的顆粒狀複合物中。此磷酸化主要是經由p38MAPK驅動之細胞訊息所調控。實驗結果顯示，RBM4會藉由辨認以及結合在核酸序列中多嘧啶的區域，進而抑制其蛋白質轉譯的活性。另外，RBM4會促進RNA解螺旋酶eIF4A與具有IRES之mRNA結合，促進其蛋白質產生的活性。此種含有IRES之mRNA多在細胞位於壓力環境下時表現以維持正常之細胞功能或驅使細胞死亡。經由此實驗結果發現RBM4在細胞質中具有調控某些mRNA產生蛋白質之活性。 RBM4除了在砷化物刺激下，同時在進行的肌肉母細胞中也會經由磷酸化而累積在細胞質中。RBM4不只負責調控α-TM在肌肉分化時進行替代性剪接以產生骨骼肌特有之形式，同時會與在肌肉分化時期表現之微小RNA(microRNA)，以及內水解酶Ago2共同參與蛋白質轉譯之抑制，藉此進一步促進肌肉母細胞之分化。綜合以上之實驗結果可知，RBM4在不同細胞及不同分化時期時，具有多種截然不同之生物活性。

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.