Molecular Studies of Nuclear Localization Signal by RNA Interference

• Main Authors: Chiao-Fang Teng, 鄧喬方
• Other Authors: Wen-Tsan Chang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/70314090739806692509

碩士 === 國立成功大學 === 生物化學研究所 === 92 === Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity. Transport of macromolecules into and out of the nucleus is generally controlled by targeting signals that are recognized by specific members of the importin/exportin transport receptor family. Several distinct types of nuclear localization signals (NLSs), from SV40 large T-antigen, nucleoplasmin, hnRNP A1, and p53, mediate passage of cargo proteins into the nucleus. RNA interference (RNAi) has shown great potential for use as a tool for biological discovery, analysis and therapeutics. In this study, we try to dissect the relationship between NLSs and nuclear transport receptors by using RNAi-based techniques. Until now, we have established the expression vectors of NLSs, including SV40 large T-antigen, nucleoplasmin, hnRNP A1, and p53, fused with one or two copies of enhanced green fluorescent protein (EGFP; EGFP2), or two copies EGFP plus glutathione S-transferase (EGFP2-GST). Simultaneously, we have screened the effective siRNAs/shRNAs against nuclear transport receptors, including human importin alpha (1,3,4,5,6,7), beta (1,2,3) family and Ran. By co-transfection of those NLS-expression vectors and shRNAs against nuclear transport receptors, we found that the shRNAs against importin beta family and Ran contrast to alpha have larger effect on the efficacy of NLS-mediated nuclear import. In particular, we were surprised to find that shRNAs against importin beta 1 and Ran individually have most dramatic effect on the efficacy of p53-NLS1-mediated nuclear import. Importantly, this result could be further observed not only by co-transfection of p53 gene-expression vector and these shRNAs but also on the endogenous level by using immunofluorescence staining. In another study, we used several shRNAs against cell survival genes, including Bcl-2, survivin, Akt1, Erk2, cyclinE, NFκB for combination and found that the combination of at most six shRNAs would not affect their silencing efficiency. Furthermore, when these shRNA-expression vectors were transfected into HeLa cell line, we interestingly observed that these shRNAs would induce different level of apoptosis. Because each type of NLS was recognized more than one type of nuclear transport receptors, this combination RNAi expression system was used to further analyze the nuclear localization mechanism by combining those shRNAs against nuclear transport receptors. Until now, we have established all possible types of combination expression vectors from those shRNAs against importin beta family and Ran, and these shRNA combination expression vectors can provide further analysis for nuclear transport mechanism in the future.