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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 poly（A）尾存在于几乎所有mRNA和许多初级microRNA转录物的3'末端，并且其影响RNA代谢的许多方面。虽然许多必需的哺乳动物3'加工因子已通过常规的生化分析，3'加工复合物的完整蛋白质组成尚未被表征，并且该过程的调节仍然知之甚少。本研究采用多维蛋白质鉴定技术（MudPIT）分离纯化了人3'加工复合物，并测定了其蛋白质组成。除了已知的3'加工因子外，我们的复合物还含有酵母3'加工因子的同源物，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、翻译和可能的Ca 2+信号传导联系起来的蛋白质。最令人惊讶的是，许多具有DNA修复功能的因子被鉴定出来。这一观察揭示了3'加工和转录之间的意想不到的相似性，其中已知几种DNA修复因子发挥重要作用。最后，我们验证了本研究中确定的几个因素在3'加工中的参与。

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.