PROTEOMIC ANALYSIS OF THE EUKARYOTIC PRE-MRNA 3' PROCESSING COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在几乎所有的mRNA和许多初级microRNA转录本的3‘端都发现了聚(A)尾巴，它影响着RNA代谢的许多方面。虽然通过常规的生化分析已经鉴定了许多哺乳动物3‘端加工因子，但3’端加工复合体的完整蛋白质组成还没有得到表征，对这一过程的调控仍然知之甚少。在本研究中，我们分离纯化了高纯度的人3‘加工复合体，并利用多维蛋白质鉴定技术(MudPIT)测定了它们的蛋白质组成。除了已知的3‘端加工因子，我们的复合体还包含酵母3’端加工因子的同系物，以及可能将3‘加工与转录、染色质重塑、剪接、RNA质量控制、输出、翻译以及可能的钙信号联系起来的蛋白质。最令人惊讶的是，发现了许多在DNA修复中起作用的因子。这一观察揭示了3‘处理和转录之间出人意料的相似之处，已知几个DNA修复因子在其中发挥关键作用。最后，我们验证了本研究中确定的几个因素参与了3‘加工。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A poly(A) tail is found at the 3' ends of nearly every mRNA and many primary microRNA transcripts, and it influences many aspects of RNA metabolism. Although many essential mammalian 3' processing factors have been identified through conventional biochemical analysis, the full protein composition of the 3' processing complex has not been characterized, and the regulation of this process is still poorly understood. In this study, we isolated highly purified human 3' processing complexes and determined their protein composition by using multidimensional protein identification technology (MudPIT). In addition to known 3' processing factors, our complex contained homologues of yeast 3' processing factors, and proteins that may link 3' processing to transcription, chromatin remodeling, splicing, RNA quality control, export, translation, and possibly Ca2+ signaling. Most surprisingly, many factors with functions in DNA repair were identified. This observation reveals an unexpected similarity between 3' processing and transcription, in which several DNA repair factors are known to play essential roles. Finally, we validated the involvement of several factors identified in this study in 3' processing.