人组织型纤溶酶原激活剂(t-PA)mRNA5′端非翻译区(UTR)对其表达的调控

Influence of 5'-Untranslated Region (5'-UTR) on the Regulation of Human Tissue-type Plasminogen Activator (t-PA) mRNA Expression

运用反转录-PCR技术,从黑色素瘤细胞中扩增出t—PA cDNA 5′末端460bp的片段,再经重组获得含完整5′-UTR的t—PA cDNA克隆,在兔网织红细胞裂解物中翻译和COS-7细胞中表达发现,t—PA mRNA 5′—UTR对其表达有明显的抑制作用。将t—PA mRNA 5′—UTR用苜蓿病毒RNA 5′—UTR替换,使t—PA的表达水平提高3-7倍,mRNA翻译起始区二级结构分析结果表明,翻译起始区的二级结构与t-PA的表达水平有关。

To investigate the influence of 5'-UTR of t-PA mRNA on its expression, first we successfully cloned t-PA 5' UTR sequence from melanoma cells. Cellular RNA was extracted from PMA induced melanoma cells, and first strand cDNA was synthesized by reverse transcription, which was used as template for polymerase chain reaction. A 460 bp fragment of t PA cDNA 5' end was obtained by PCR. Sequence analysis showed that the amplified sequence was identical to that of the first exon of t-PA gene previously reported. Full lengths t-PA cDNA clone was obtained by DNA recombination. Inhibitory effect of t PA 5' UTR on its expression was observed both in RRL and in COS 7 cells. Then t- PA mRNA 5' UTR was replaced by that of AMV RNA, a very efficiently translated RNA. The substitution resulted in 3-7 fold increase of t-PA expression in COS-7 cells. Computer prediction of secondary structure of mRNA translation initiation region showed that the stem-loop structure is most stable for t PA native mRNA, and less stable when 5'-UTR was deleted, and most unstable for AMV-t-PA chimeric mRNA. The stability of secondary structure in translation initiation region might be related to t-PA expression. Our results, together with previously reported, demonstrated that 5'-UTR derived from efficiently translated mRNAs may enhance the expression of other coding sequences when presented in cis, which suggested that consideration of the structure and sequence of mRNA 5'-UTR should be an important aspect of efficient expression for foreign genes in eukaryotic cells.