Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription.
Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription.
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DOI：
10.1101/gr.5211806
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发表时间：
2006-07
期刊：
影响因子：
7
通讯作者：
Ian A. Swinburne;Clifford A. Meyer;X. Liu;P. Silver;A. Brodsky
中科院分区：
文献类型：
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作者：
Ian A. Swinburne;Clifford A. Meyer;X. Liu;P. Silver;A. Brodsky
前体mRNA加工通常与转录协同发生，从而将这两个关键的调控事件偶联起来。因此，许多参与mRNA加工的蛋白质与转录机器相关联，并靠近DNA。这种靠近使得通过染色质免疫沉淀（ChIP）绘制RNA结合蛋白的基因组关联图谱成为可能，以此确定它们在编码mRNA上的作用位点。在此，我们利用ChIP结合高密度微阵列在人类基因组上定位三种功能不同的RNA结合蛋白：剪接因子多聚嘧啶区结合蛋白（PTBP1/hnRNP I）、mRNA输出因子THO复合物亚基4（ALY/THOC4）以及3'端切割刺激因子64 kDa（CSTF2）。我们观察到在活跃基因的启动子、内部外显子和3'端存在相互作用。PTBP1倾向于启动子，并且常常与RNA聚合酶II（RNA Pol II）重合。3'加工因子CSTF2倾向于3'端，但也在启动子处被观察到。mRNA加工和输出因子ALY定位到一些外显子，但主要位于内含子，且不与RNA Pol II重合。由于RNA结合蛋白并不总是与RNA Pol II重合，这些数据支持一种由转录复合物重组驱动的加工机制，而非扫描机制。总之，我们展示了在哺乳动物细胞中RNA结合蛋白在部分基因组上的定位图谱，这为RNA - 蛋白质复合物的转录组装提供了见解。
Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3' end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3' ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3' processing factor, CSTF2, had biases toward 3' ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA-protein complexes.