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Poly(A)尾巴，它影响着许多 RNA新陈代谢的各个方面。尽管许多重要的哺乳动物3‘加工因子已经通过 常规生化分析，尚未表征3‘加工复合体的全部蛋白质组成， 对这一过程的监管仍然知之甚少。在这项研究中，我们分离出高纯度的人类3‘加工 并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‘加工因子，我们的复合体还含有酵母3’加工因子的同系物，以及蛋白质 这可能将3‘处理与转录、染色质重塑、剪接、RNA质量控制、输出、翻译和 可能是钙离子信号。最令人惊讶的是，发现了许多在DNA修复中起作用的因子。这一观察结果 揭示了3‘处理和转录之间出人意料的相似之处，其中几个DNA修复因子已知 扮演着至关重要的角色。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.