Tristetraprolin recruits functional mRNA decay complexes to ARE sequences
Tristetraprolin recruits functional mRNA decay complexes to ARE sequences
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DOI：
10.1002/jcb.21130
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发表时间：
2007-04-15
影响因子：
4
通讯作者：
Bohjanen, Paul R.
中科院分区：
文献类型：
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作者：
Hau, Heidi H.;Walsh, Richard J.;Bohjanen, Paul R.
在许多哺乳动物转录物的3'非翻译区（UTR）中的富含AU的元件（战神）作为促进mRNA快速降解的不稳定元件发挥作用。Tristetraprolin（TTP）是一种ARE结合蛋白，通过尚不清楚的机制促进mRNA快速衰减。来自TNF-α 3' UTR的31个核小体ARE序列在插入β-珠蛋白报告转录物的3' UTR中时促进TTP依赖性mRNA衰变，表明该短序列足以实现TTP功能。我们使用凝胶迁移试验，以确定从TTP转染的HeLa细胞的细胞质提取物中含有TTP的复合物，特异性结合短ARE序列。该含TTP的复合物还含有5 '-3'核酸外切酶Xrn 1和外泌体组分PM-scl 75，因为它被抗Xrn 1或抗PMscl 75抗体超移位。RNA亲和纯化证实，这些蛋白与ARE序列特异性相关的TTP依赖的方式。使用竞争结合测定，我们发现含TTP的复合物以高亲和力结合来自GM-CSF、IL-3、TNF-α、IL-2和c-fos的短ARE序列，但不结合来自c-myc的富含U的序列、22个核苷酸的多聚U序列或突变的GM-CSF控制序列。在无细胞mRNA衰变试验中，含TTP复合物的高亲和力结合与TTP依赖性去腺苷酸化和加帽多腺苷酸化转录物的衰变相关，表明含TTP复合物具有功能。这些数据支持TTP通过将细胞mRNA衰变机制的组分募集到转录物中来增强mRNA衰变的模型。
AU-rich elements (AREs) in the 3' untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleoticle ARE sequences from the TNF-alpha 3' UTR promoted TTP-dependent mRNA decay when it was inserted into the 3' UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5'-3' exonuclease Xrn 1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript.