Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster
Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster
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DOI：
10.1073/pnas.0509260103
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发表时间：
2006-03-21
影响因子：
11.1
通讯作者：
Herschlag, D
中科院分区：
文献类型：
--
作者：
Gerber, AP;Luschnig, S;Herschlag, D
全基因组鉴定与rna结合蛋白相关的rna对于破译转录后调控系统至关重要。PUMILIO是进化保守的rna结合蛋白puf家族的一员，该家族在转录后抑制基因表达。我们在卵巢特异性启动子的控制下产生了表达亲和标记PUMILIO的转基因果蝇，并从整个成年果蝇和胚胎中纯化了PUMILIO，并使用DNA微阵列分析了相关mrna。包括数百个mrna的不同集合在两个发育阶段与PUMILIO相关。许多这些mrna编码功能相关蛋白，支持特定rna结合蛋白协调调节转录后模块的模型。我们在与PUMILIO相关的mrna的3'-非翻译区发现了一个特征序列基序，并通过生物素化合成RNA的RNA拉下实验证实了该基序与PUMILIO相互作用的充分性。该RNA基序与先前鉴定的Puf3p（酿酒酵母菌Puf蛋白的五种之一）非常相似；然而，酵母和果蝇中相关mrna编码的蛋白质似乎并不相关。结果表明PUMILIO具有广泛的转录后调控作用，并揭示了这一保守的rna结合蛋白家族的进化特征。
Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.