Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors
Genome-wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors
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DOI：
10.1186/gb-2006-7-11-r113
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发表时间：
2006-01-01
期刊：
影响因子：
12.3
通讯作者：
Carmo-Fonseca, Maria
中科院分区：
文献类型：
--
作者：
Gama-Carvalho, Margarida;Barbosa-Morais, Nuno L.;Carmo-Fonseca, Maria
背景：前体信使核糖核酸（pre - mRNA）剪接是基因表达的一个关键步骤，在细胞核内与转录同时发生，除了核心剪接体成分外，还涉及大量的核糖核酸结合蛋白剪接因子。其中一些蛋白质是识别内含子序列元件所必需的，在剪接过程中与初级转录本短暂结合。一些蛋白质剪接因子，比如U2小核核糖核蛋白辅助因子（U2AF），尽管仅涉及核功能，但已知会被输出到细胞质。这一观察结果引发了一个问题，即U2AF是否会在运往细胞质的过程中与成熟的信使核糖核酸 - 核糖核蛋白颗粒结合，从而参与其他细胞功能。
结果：在此我们报道了由一种针对U2AF 65 kDa亚基（U2AF(65)）的单克隆抗体免疫沉淀的核糖核酸的鉴定，并证明了它与剪接后的信使核糖核酸的结合。为了进行比较，我们分析了与多聚嘧啶区结合蛋白（PTB）相关的信使核糖核酸，PTB是一种剪接因子，它也与内含子中富含嘧啶的序列结合，但还参与信使核糖核酸的定位、稳定性和翻译。我们的结果显示，在海拉细胞中表达的10%的细胞信使核糖核酸与U2AF(65)和PTB有差异结合。在与U2AF(65)相关的信使核糖核酸中，转录因子和细胞周期调节因子占主导，而与PTB相关的转录本在编码与细胞内运输、囊泡运输和细胞凋亡有关的蛋白质的信使核糖核酸种类中富集。
结论：我们的结果表明，U2AF(65)与剪接后的信使核糖核酸的特定子集结合，有力地表明它除了参与剪接外，还涉及新的细胞功能。
Background: Pre-mRNA splicing is an essential step in gene expression that occurs cotranscriptionally in the cell nucleus, involving a large number of RNA binding protein splicing factors, in addition to core spliceosome components. Several of these proteins are required for the recognition of intronic sequence elements, transiently associating with the primary transcript during splicing. Some protein splicing factors, such as the U2 small nuclear RNP auxiliary factor (U2AF), are known to be exported to the cytoplasm, despite being implicated solely in nuclear functions. This observation raises the question of whether U2AF associates with mature mRNA-ribonucleoprotein particles in transit to the cytoplasm, participating in additional cellular functions.Results: Here we report the identification of RNAs immunoprecipitated by a monoclonal antibody specific for the U2AF 65 kDa subunit (U2AF(65)) and demonstrate its association with spliced mRNAs. For comparison, we analyzed mRNAs associated with the polypyrimidine tract binding protein (PTB), a splicing factor that also binds to intronic pyrimidine-rich sequences but additionally participates in mRNA localization, stability, and translation. Our results show that 10% of cellular mRNAs expressed in HeLa cells associate differentially with U2AF(65) and PTB. Among U2AF(65)associated mRNAs there is a predominance of transcription factors and cell cycle regulators, whereas PTB-associated transcripts are enriched in mRNA species that encode proteins implicated in intracellular transport, vesicle trafficking, and apoptosis.Conclusion: Our results show that U2AF(65) associates with specific subsets of spliced mRNAs, strongly suggesting that it is involved in novel cellular functions in addition to splicing.