Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.
J Thromb Haemost. 2012 May;10(5):887-94. doi: 10.1111/j.1538-7836.2012.04689.x.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase zymogen encoded by the human gene CPB2. TAFI constitutes a molecular link between coagulation and fibrinolysis, and between coagulation and inflammation. The 3'-untranslated region (UTR) of the human CPB2 mRNA plays a key role in regulating CPB2 mRNA abundance, but the exact mechanisms that mediate this regulation are largely unexplored.
To pinpoint cis-acting elements in the CPB2 3'-UTR that act as stability determinants and to identify protein factors binding to these sites.
We constructed a series of plasmids encoding mRNAs containing rabbit β-globin sequences (as a reporter) fused to sequences of the CPB2 3'-UTR (encompassing 5' and internal deletions). These plasmids were transfected into HepG2 (human hepatoma) cells and the stability of the fusion transcripts measured. We performed a series of gel mobility shift analyses using RNA probes encompassing putative (in)stability elements.
We identified one element conferring stability and three elements conferring instability. Supershift assays identified the protein bound to the site between the second and third polyadenylation sites as tristetraprolin (TTP). Mutation of the TTP site abolished TTP binding in gel mobility shift assays and also stabilized β-globin/CPB2 fusion transcripts. TTP knockdown stabilized the fusion transcript containing the TTP site, but not a fusion transcript in which this site was mutated.
Our findings are indicative of a role for TTP in constitutive, and perhaps regulated, control of CPB2 mRNA stability and hence abundance.
凝血酶激活的纤溶抑制物（TAFI）是一种由人类基因 CPB2 编码的基本羧肽酶原。TAFI 构成了凝血和纤溶之间以及凝血和炎症之间的分子联系。人类 CPB2 mRNA 的 3'-非翻译区（UTR）在调节 CPB2 mRNA 丰度方面起着关键作用，但介导这种调节的确切机制在很大程度上尚未探索。
确定 CPB2 3'-UTR 中作为稳定性决定因素的顺式作用元件，并鉴定与这些位点结合的蛋白质因子。
我们构建了一系列质粒，这些质粒编码的 mRNA 包含兔珠蛋白序列（作为报告基因）与 CPB2 3'-UTR 的序列融合（包含 5'和内部缺失）。这些质粒被转染到 HepG2（人肝癌）细胞中，并测量融合转录物的稳定性。我们使用包含潜在（不稳定）元件的 RNA 探针进行了一系列凝胶迁移率变动分析。
我们确定了一个赋予稳定性的元件和三个赋予不稳定性的元件。超迁移分析鉴定出与第二个和第三个多聚腺苷酸化位点之间的位点结合的蛋白质为三肽重复蛋白（TTP）。TTP 位点的突变消除了凝胶迁移率变动分析中的 TTP 结合，并且还稳定了β-球蛋白/CPB2 融合转录物。TTP 敲低稳定了包含 TTP 位点的融合转录物，但不能稳定该位点发生突变的融合转录物。
我们的发现表明 TTP 在 CPB2 mRNA 稳定性和丰度的组成型（或许受调控）控制中起作用。
