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研究生:林榮俊
研究生(外文):Jung-Chun Lin
論文名稱:探討mRNA前驅分子之剪接調控蛋白RBM4於細胞內的功能
論文名稱(外文):Functional study of pre-mRNA splicing regulator RBM4
指導教授:譚婉玉
指導教授(外文):Woan-Yuh Tarn
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
中文關鍵詞:核糖核酸蛋白複合物分示法替代性剪接
外文關鍵詞:alternative splicingRibonucleoproteinsRBM4differential displaytropomyosin
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訊息RNA (mRNA) 會藉由不同的剪接(alternative splicing) 方式來造
成蛋白質的多樣性,這種現象可在不同的組織中或發育時期受到嚴密的調控以符
合生理功能。細胞中的mRNA 多會形成巨大的核糖核酸蛋白複合物
(Ribonucleoproteins; RNPs)。這些RNP 蛋白可負責不同的功能,例如mRNA
的修飾、運輸或定位。蛋白質進出細胞核則需藉助於運輸蛋白的參與。我們經由
酵母菌雙雜合篩選法(yeast two-hybrid screen) 篩選出一個具有RNA recognition
motif (RRM) 的蛋白質,名為有氧核醣核酸附著蛋白(RNA binding protein
motif protein 4;RBM4) 。RBM4 是一個RNA 的剪接因子(splicing factor),
並且具有RNA 結合區域,但至於RBM4 是否會參與特定mRNA 的調控仍然
不清楚。本篇論文的研究目的即是探討RBM4 是否會和某一群特定的mRNA
形成RNP 複合物,以及對於此RNA 是否具有特定的功能。藉由RT-PCR 的方
法我們得知RBM4 在大多數的組織中都會表現,但是在橫紋肌以及心肌中的含
量遠高於其他組織。我們利用免疫沉澱和分示法(differential display)的方法來檢
視會和RBM4 形成複合物的mRNA,截至目前我們找到了16 種特定的
RNA ,其中4 種所產生的蛋白質會和actin 結合在一起負責形成細胞骨架以及
調控肌肉細胞的收縮,於是我們推測RBM4 在肌肉細胞中對這些RNAs 可能
具有轉錄後的調節功能。α-tropomyosin mRNA 是會和RBM4 結合的RNA
之一,此RNA 在橫紋肌和平滑肌中存在著不同的splicing patterns。藉由定量
RT-PCR 的實驗我們發現當RBM4 在細胞中被大量表現時,會使得骨骼肌特有
的splicing pattern 也同時增加。將α-TM 的迷你基因(mini gene)和RBM4 同
時送到細胞中,也可以觀察到相同的情形。因此我們推論RBM4 可能調控了
α-TM 在肌肉細胞中的替代性剪接(alternative splicing),以產生具有符合肌細胞
生理功能的蛋白質形式。
1
Alternative splicing of pre-mRNA is regulated by various RNA binding proteins.
We previously identified a novel RNA-binding protein, RBM4, that can determine
splice site selection of pre-mRNA. To elucidate the cellular function of RBM4, we
attempt to search for the mRNA targets and interacting partners of RBM4. The object
of the thesis is to investigate whether RBM4 recognizes specific mRNAs and
participates in their post-transcriptional regulation. First, we found that RBM4 is
associated with mRNA in RNP complexes. Next, we used a ribonomic approach to
identify mRNAs that are specifically associated with RBM4. The result showed that
RBM4 may bind to a set of mRNAs encoding muscle-specific or cytoskeletal proteins,
including α-tropomyosin (α-TM). This observation correlates well with the tissue
specificity of RBM4 and suggests that RBM4 is involved in muscle-specific
post-transcriptional gene regulation. Ectopic expression of RBM4 promotes the
skeletal muscle-specific alternative splicing of α-TM in HEK293 cells. Using a
minigene construct containing α-TM terminal exons, we found that overexpression of
RBM4 activated skeletal muscle-specific splicing of α-TM. Moreover, we
demonstrated that RBM4 can suppress skipping of exon 3 of α-TM, consistent with its
potential role in regulation of muscle-specific splicing. The possibility of whether
RBM4 is also involved in other post-transcriptional regulation events of
muscle-specific genes will be examined in the near future.
66
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