Influence of nonameric AU-rich tristetraprolin-binding sites on mRNA deadenylation and turnover
Influence of nonameric AU-rich tristetraprolin-binding sites on mRNA deadenylation and turnover
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DOI：
10.1074/jbc.m506757200
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发表时间：
2005-10-07
影响因子：
4.8
通讯作者：
Blackshear, PJ
中科院分区：
文献类型：
--
作者：
Lai, WS;Carrick, DM;Blackshear, PJ
tristetrprolin （TTP）是CCCH锌指蛋白家族的一员，在与富au元件（ARE）的3'-非翻译区结合后，可促进肿瘤坏死因子- α和粒细胞-巨噬细胞集落刺激因子mrna的死烯化。高亲和性的TTP-ARE结合发生在串联锌指结构域和首选命名物UUAUUUAUU之间。通过突变肿瘤坏死因子- α ARE中一个明确定义的24个碱基核心序列，我们比较了野生型ARE中四个可能的非美性TTP结合位点与突变探针中单个结合位点对TTP与RNA结合以及随后聚(a)尾部死基化的影响。通过将这个24碱基的ARE插入到一个稳定的转录本中，我们还试图确定一个或两个ttp结合位点的存在所带来的不稳定性的程度。这些位点是通过改变UUAUUUAUU氨基中的As或改变氨基的中心U来产生或修改的，在这两种情况下都是C残基。结果表明，即使是单一的非聚合体ttp结合位点，也可以对ttp介导的mRNA在其他稳定的mRNA上的转换至少部分敏感，但两个结合位点使转录物更加不稳定。尽管结构研究预测非聚合体结合位点的中心U可能允许碱基置换，但该U到C的突变极大地抑制了TTP与ARE的结合，从而降低了TTP促进死基化和mRNA不稳定性的能力。
Tristetraprolin (TTP), a member of the tandem CCCH zinc finger protein family, promotes deadenylation of tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to the AU-rich elements ( ARE) in their 3'-untranslated regions. The high affinity TTP-ARE binding occurs between the tandem zinc finger domain and the preferred nonamer UUAUUUAUU. By mutating a well defined core sequence of 24 bases from the tumor necrosis factor-alpha ARE, we compared the influence of four possible nonameric TTP-binding sites in the wildtype ARE with that of a single binding site in the mutated probe on the binding of TTP to the RNA and the subsequent deadenylation of the poly( A) tail. By inserting this 24-base ARE into an otherwise stable transcript, we also attempted to determine the extent of the instability conferred by the presence of one or two TTP-binding sites. These sites were created or modified by mutating the As in the UUAUUUAUU nonamer or by changing the central U in the nonamer, in both cases to C residues. The results suggest that even a single nonamer TTP-binding site can confer at least partial sensitivity to the TTP-mediated mRNA turnover on an otherwise stable mRNA, but that two binding sites make the transcript much more unstable. Even though the central U of the nonamer binding site was predicted by structural studies possibly to permit base substitution, mutation of this U to C greatly inhibited the binding of TTP to the ARE, thus reducing the ability of the TTP to promote deadenylation and instability of the mRNA.