Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs
Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs
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DOI：
10.1093/nar/27.6.1464
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发表时间：
1999-03-15
影响因子：
14.9
通讯作者：
Malter, JS
中科院分区：
文献类型：
--
作者：
Bhattacharya, S;Giordano, T;Malter, JS
细胞激活与转录和转录后水平上基因表达的多样化和广泛变化相关。 AUF1 是最近描述的一种细胞质蛋白，它可能参与富含 AU (ARE) mRNA 的转录后调节 (PTR)，包括那些编码细胞因子和原癌基因的 mRNA。如果 ARE 存在或者所讨论的 mRNA 在体外或体内与 AUF1 相互作用，则可以预测受 AUF1 介导的 PTR 影响的单个 mRNA。然而，很少有通用方法来表征受 AUF1 PTR 影响的 mRNA 的整体库，为了鉴定这些 mRNA，我们在体外将丝裂原激活的外周血单核细胞 (PBMC) 的总 mRNA 与 AUF1 一起孵育，AUF1-mRNA 复合物在膜上被阻滞，结合的 mRNA 用高盐洗脱，然后用于生成 cDNA 文库或在第二次或第三次反弹至 AUF1洗脱和 cDNA 文库构建之前的时间。我们从130个克隆中获得了部分核苷酸序列，这表明AUF1选择的文库富含含有3'非翻译区ARE的mRNA，包括大量早期反应基因cDNA。作为对该方法有效性的测试，我们还表明文库中包含的随机选择的新型mRNA在细胞激活后是稳定的。
Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes, Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1, In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mononuclear cells (PBMCs) with AUF1 in vitro, AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs, As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.