Cef1p/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 Cef1p/Ntc85p的功能相似體。所以像Cef1p/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Liver cDNA基因庫的篩選，篩選了3*10/sup 5/個質體，目前篩選出12個菌落，其中並沒有基因跟Pre-mRNA剪接反應有直接關聯，但是有3個基因是轉錄因子(Transcription factor): Prox1、C/EBPδ與HARS1。5個基因與蛋白運送有關，4個基因功能未知。經由酵母菌Two-hybrid分析，證實hCDC5p與3個轉錄因子：Prox1、C/EBPδ與HARS1確實可以特定性結合；同時證實C/EBPδ和HARS1的Leucine dimerization domain是負責與hCDC5p的結合。這些結果與cDNA基因庫的篩選是相似的。所以hCDC5p和這些與hCDC5p結合的轉錄因子將是研究基因的轉錄與剪接反應彼此如何關聯的有利工具。 The Cef1p/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 Cef1p/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. 3x10/sup 5/ clones were screened from human liver cDNA library. So far, 12 clones were isolated. No clone has the known function related to pre-mRNA splicing. However, three transcription factors, Prox1, C/EBPδ, and HARS1, were isolated, five clones are related to the function for protein translocation across the nuclear membrane and four clones have the unknown function. Full length of Prox1p interact with hCDC5p, however, full length of C/EBPδ and HARS1 have very weak interaction with hCDC5p by yeast two-hybrid assay. Different truncated proteins of C/EBPδ and HARS1 were constructed and leucine dimerization domain in both proteins has clear interaction with hCDC5p. These results are similar with those in the liver library screening. Therefore, these hCDC5p-interacting transcription factors will be useful tools to study the coupling mechanism between the transcription and the pre-mRNA splicing of genes.
