在体和全转录组鉴定 RNA 结合蛋白靶位。

In vivo and transcriptome-wide identification of RNA binding protein target sites.

机构信息

Systems Biology of Gene Regulatory Elements, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

出版信息

Mol Cell. 2011 Dec 9;44(5):828-40. doi: 10.1016/j.molcel.2011.11.009.

Abstract

Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes.

摘要

动物 mRNA 受到数百种 RNA 结合蛋白 (RBP) 的调控。鉴定 RBP 的靶标对于理解其功能至关重要。最近的一种方法,PAR-CLIP,使用光反应性核苷在免疫沉淀之前将 RBP 交联到细胞中的靶 RNA 上。在这里,我们建立了 iPAR-CLIP(体内 PAR-CLIP),以确定 GLD-1 的全转录组结合位点,GLD-1 是秀丽隐杆线虫中一种保守的、生殖细胞特异性的翻译抑制剂。我们鉴定了 439 个可重复的靶 mRNA,并通过 iPAR-CLIP 证明了靶标检测的出色动态范围。在 GLD-1 敲低后,439 个靶标的蛋白质而非 mRNA 表达被特异性地上调,表明其具有功能。最后,我们在靶基因的起始密码子附近发现了强烈保守的 GLD-1 结合位点。这些位点在体外具有功能,并且可能在体内具有很强的抑制作用。我们提出 GLD-1 在起始密码子附近与翻译机制相互作用,这是真核生物中一种迄今为止未知的基因调控模式。

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