細胞在對於DNA轉錄成RNA，到RNA轉譯成蛋白質的最終生成量上有著多重的調控機制，其中在RNA的層級上，可以經由不同的cis-acting elements及trans-acting factors來調控RNA的穩定度及RNA在轉譯時的效率，進而影響蛋白質最終的合成量。IRES是其中一種調控轉譯的cis-acting element，其位於mRNA的5’端非轉譯區(5’UTR)，具有直接招聚核醣體(Ribosomes)的能力，因而提供一條不須經由cap即可進行轉譯啟始的途徑。HuR是一個廣泛存在於生物體中的RNA結合蛋白，其被廣為人知的功能是可以藉由與帶有AU-rich的mRNA 3’UTR結合，增強目標mRNA的穩定度。由於HuR能夠調節mRNA的出核、穩定度與轉譯，因此在後轉錄修飾的調控(post-transcriptional regulation) 扮演了一個重要的角色。先前的研究發現，在IL-1β的刺激下HuR可以增加與凝血酶調節素( thrombomodulin，TM ) mRNA 5’端非轉譯區作用。TM在抗凝血及抗發炎中扮演著調節的角色。此外，我們亦發現HuR還可以與TM mRNA的3’端非轉譯區作用而增加TM mRNA的穩定度。
由於HuR可以同時與TM mRNA的3’及5’UTR作用，並達到各自的調控，因此我們想要進一步去探討HuR如何分別去調控這兩個非轉譯區。 HuR蛋白上面帶有三個RNA recognition motifs ( RRMs)，首先利用了biotin pull-down分析，確認了HuR經由第三個RRM (RRM3)與TM 5’UTR作用，而第一個RRM (RRM1)及第二個RRM (RRM2)則在HuR與TM 3’UTR的結合上扮演了角色。在RRM1及RRM2的定點突變株(site-directed mutants) ( F65/Y68A及F154/157A )，同樣也觀察到這兩株突變株無法與TM 3’UTR結合。此外，reporter assay的結果顯示，當HuR同時與TM 5’及3’端非轉譯區結合時，HuR對TM轉譯抑制的效果會比單獨結合到TM 5’端非轉譯區的抑制效果還要明顯。此外，我們發現了另一個RNA結合蛋白polypyrimidine-tract-binding protein (PTB)也會參與在TM的後轉錄調控當中，其可能與HuR互相競爭，能夠促進TM的表現。
總結來說，HuR藉由RRM3與TM 5’端非轉譯區作用，而藉由RRM1及RRM2與TM 3’端結合，當HuR能夠同時與TM mRNA的兩端結合時，能提供HuR較佳的作用效率。此外，PTB能藉由參與在TM的後轉錄調控而促進TM的表現。

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.

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