To study the molecular mechanism of HuR mediating TMprotein expression

• Main Authors: Li-FangChen, 陳俐方
• Other Authors: Joseph T. Tseng
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/53684444989492113795

碩士 === 國立成功大學 === 生物資訊研究所 === 98 === The cell has many ways to regulate the production of proteins, including the modulation of translation efficiency and transcripts turnover through cis-acting elements and trans-acting factors. One of the cis-acting elements is IRES (internal ribosomal entry site). IRES is a RNA element that can recruit ribosome complex and allow cap-independent translation initiation in the mRNA 5’UTR. HuR is a ubiquitous RNA-binding protein which is well-known to stabilize mRNA by interacting with AU-rich element-containing mRNA and plays an important role in post-transcriptional regulation through altered mRNA export, turnover and translation. Our previous study demonstrates that HuR interacts with the 5’UTR of thrombomodulin (TM), an critical factor in mediating anticoagulation and anti-inflammation, and represses TM IRES activity under IL-1β treatment. Besides, there is also an AU-rich element in TM mRNA 3’UTR, and HuR can also stabilize TM mRNA via interacting with TM mRNA 3’UTR. Since HuR may regulate TM expression by simultaneous interacting with TM mRNA 5’and 3’ UTRs, we are interesting to explore how HuR coordinates these two elements to regulate TM expression. According to the result of biotin pull-down assay, we find that HuR RNA recognition motif (RRM) 1 and 2 are important for the direct interaction between HuR and TM 3’UTR; meanwhile, HuR RRM 3 is related to interact with TM 5’ UTR. Site direct mutagenesis of F65/Y68A of RRM1 and F154/157A of RRM2 will abolish the binding of HuR with TM 3’UTR. Furthermore, we demonstrate that HuR simultaneously bound with 3’UTR and 5’UTR displays higher repression effect on TM translation than bound with 5’UTR only by reporter assay. Besides, we find that polypyrimidine-tract-binding protein (PTB), another RNA-binding protein, also involved in the translation regulation of TM protein expression. In conclusion, HuR interact with TM 5’UTR via RRM3 and with TM 3’UTR by RRM1 and RRM2. It provides better repression effect on TM IRES when HuR simultaneously binds with TM 5’and 3’UTR. Besides, PTB also play a role in enhancing TM protein expression.