真核細胞的剪接體(Spliceosome)是經過一系列有次序性步驟的過程，組合而成的核醣核酸蛋白複合體(Ribonucleoprotein particle)；而pre-mRNA剪接反應(Splicing reaction)是發生在剪接體上，經過兩步驟催化反應的過程。Ceflp/Ntc85p是酵母菌Saccharomyces cerevisiae的一個必需剪接蛋白(Splicing factor)，同時會形成一個至少含有8個蛋白質的Prp19p複合體。人類hCDC5p與酵母菌Schizosaccharomyces pombe CDC5p是酵母菌S. cerevisiae Ceflp/Ntc85p的功能相似體。所以像Ceflp/Ntc85p，hCDC5p與CDC5p也都是pre-mRNA剪接蛋白；另外CDC5p與hCDC5p也會形成巨大的蛋白複合體。因此經由酵母菌Two-hybrid分析，我們利用hCDC5p當作探針，可以來尋找hCDC5p的結合蛋白。我們總共進行兩次HeLa cDNA基因庫的篩選，篩選了3*10/sup 5/個質體，目前我們篩選出24個菌落，其中有5個菌落有很強的藍色呈色反應，12個菌落有強的藍色呈色反應，7個菌落有弱的藍色呈色反應。有很強的藍色呈色反應的5個含基因的pACT2質體先被DNA定序，其中一個基因功能未知，四個基因有已知的功能，但是只有一個基因跟pre-mRNA剪接反應有關。這個研究計畫我們希望可以經由新的哺乳類pre-mRNA剪接蛋白的鑑定，讓我們對哺乳類pre-mRNA剪接反應有更完整的認識。 The pre-mRNA splicing reaction takes place in two catalytic steps within the spliceosome, a large multi-protein-snRNA complex that assembles in a stepwise pathway. The Ceflp/Ntc85p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the Prpl9p-associated complex consisting of at least eight protein components. Ceflp/Ntc85p is highly homologous to human hCDC5p and fission yeast Schizosaccharomyces pombe CDC5p with 48% identity. Like Ceflp/Ntc85p, human hCDC5p and S. pombe Cdc5p are also required for pre-mRNA splicing and both Ceflp/Ntc85p and Cdc5p form the similar large protein complex. Therefore, human hCDC5p will be used as a useful probe to identify novel mammalian splicing factors by yeast two-hybrid assays. 3*10/sup 5/ clones were screened from HeLa cDNA library. So far, 24 clones were isolated: 5 clones have the strong blue reaction, 12 clones have the middle blue reaction, and 7 clones have the weak blue reaction. 5 strong blue of clones were sequenced: one clone is unknown gene and four clones are known genes. However, only one clone has the known function related to pre-mRNA splicing. Finally, we hope that some novel mammalian splicing factors can be identified and act as useful tools to study the detailed mechanism of mammalian pre-mRNA splicing.
