在人類基因體中辨識微小核甘酸轉譯啟始位置

Abstract

微小核甘酸非常小，大約由21個核甘酸構成，在核甘酸干擾機制中微小核甘酸藉由完全互補或是部分互補來抑制目標基因的表現。絕大多數的微小核甘酸是由核甘酸聚合酶II來進行轉譯。經由核甘酸聚合酶II所轉譯出來的原始微小核甘酸具有5’端帽以及3’端腺嘌呤尾。在這些微小核甘酸中，有一部分的微小核甘酸基因沒有覆蓋到其他已知的基因序列上，這些微小核甘酸稱之為基因間微小核甘酸，基因間微小核甘酸具有獨立的啟動子。在研究基因間微小核甘酸的調控時，首先要知道這些基因間微小核甘酸啟動子的位置。由於傳統聚合酶連鎖反應中所使用的去氧核醣核甘酸聚合酶的酵素特性，該酵素很難完全地合成出完整的5’端片段序列。在本研究之中，我們使用了5’端迅速放大互補去氧核甘酸終端的技術來確保可以得到原始微小核甘酸的5’端序列，並藉由整合微小核甘酸的表現資料來過濾掉那些低表現量的微小核甘酸。經由一系列的電腦輔助啟動子的預測，我們可以選出那些含有各種證據所支持的可能轉錄起始位置。為了要辨認出這些可能的轉錄起始位置，我們針對這些位置設計了專一性的引子。最後再藉由反轉錄聚合酶連鎖反應來證明我們找到的可能轉錄起是位置的真偽。

The miRNAs are ~21nt single-strand short nucleotide that can induce RNAi mechanism through complete or partial complementarities. Most of the miRNAs are transcribed by RNA polymerase II [1]. The pri-miRNA transcribed by RNA polymerase II contained 5’cap and 3’poly-A tail [2]. For those intergenic miRNA, non-gene-overlap miRNA, they have their own promoter. It is important to understand those promoters of intergenic miRNA gene to facilitated understanding the regulation of intergenic miRNA. Because of the nature of the enzymatic reaction the probability of retrieving the sequence of extreme 5’ end region was very low with traditional PCR technology. Here, we use 5’RACE [3, 4] to ensure that the 5’end region of pri-miRNAs was obtained. In addition, we incorporate miRNA expression profile [5] to filter out those lower-expressed miRNA. With a series of computational promoter prediction, we could choose the putative TSS that has many support evidences. In order to identify whether putative TSS is true or false, the putative TSS specific primer was designed. Finally, the RT-PCT results were used to confirm the putative promoter regions.