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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在几乎每种mRNA和许多初级microRNA转录物的3'末端都发现了poly（A）尾，它影响了许多转录物的转录。 RNA的代谢。虽然许多重要的哺乳动物3'加工因子已被确定， 常规生化分析中，3 ′加工复合物的完整蛋白质组成尚未被表征， 并且对这一过程的调节仍然知之甚少。在这项研究中，我们分离了高度纯化的人3'加工， 利用多维蛋白质鉴定技术（MudPIT）测定了复合物的蛋白质组成。 除了已知的3'加工因子外，我们的复合物还含有酵母3'加工因子和蛋白质的同源物 它可以将3'加工与转录、染色质重塑、剪接、RNA质量控制、输出、翻译和转录联系起来， 可能是Ca 2+信号。最令人惊讶的是，许多具有DNA修复功能的因子被鉴定出来。该观察结果 揭示了3'加工和转录之间意想不到的相似性，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.