Relationship of cytoplasmic capping to post-transcriptional gene regulation

 描述(申请人提供)：这项提案涉及到前所未有的细胞质中的mRNA封顶现象。所有mRNAs的5‘端都有一个m7G’帽‘，与帽结合的蛋白质指导着每个转录本的加工、翻译和命运。流行的观点认为，帽子只能添加到细胞核中新合成的前-mRNAs中，而帽子的丧失将不可逆转地导致mRNA衰退。相反，我们鉴定了在无封顶状态下稳定的转录本，鉴定了可以将帽子恢复到这些转录本上的蛋白质的细胞质复合体，并鉴定了一个称为“帽内稳”的去帽和重排的循环过程，该过程维持了转录组的一个子集处于积极的翻译状态。细胞质封顶的过程包括将未封顶的RNA的5‘-单磷酸末端转化为5’-二磷酸，并将GMP从封端酶转移到这个重新封盖的底物上。催化细胞质封顶所需的所有酶都存在于一个单一的复合体中，该复合体组装在Nck 1上，Nck 1是一种细胞质SH2/SH3适配蛋白，最为人所知的是酪氨酸激酶信号的转导。RNA5‘-激酶和封闭酶通过与相邻的SH3结构域结合并列在一起，而复合体中CAP甲基转移酶的存在完善了影响细胞质封顶所必需的和足够的蛋白质清单。细胞质封顶靶标不是随机的；它们编码参与核苷酸结合、蛋白质定位、RNA定位和有丝分裂细胞周期的蛋白质。这一建议的工作假设是，细胞质封顶是一个选择性的转录后过程，其功能是转录组和蛋白质组复杂性的放大。在目标1中，将使用体外、体内和生化生物学方法来表征5‘-激酶及其在细胞质封顶中的功能。NKK1有4个功能结构域，经典的和“自上而下”的蛋白质组学和生化方法将被用来鉴定和鉴定在细胞质封顶复合体中唯一结合到第一个SH3结构域和SH2结构域的蛋白质。目的2将绘制重排转录本的5‘端，并确定它们与基因表达封顶分析(CAGE)确定的内部帽子位置的关系。将挖掘得到的数据集以确定序列和/或结构基序，这些基序和/或结构基序确定重排的5‘端的位置及其在确定靶标特异性中的作用。在AIM 3中，核糖体图谱将与位置蛋白质组学相结合，以确定细胞质封顶与翻译和蛋白质组复杂性的关系。这一结果将得到来自内部封顶转录本的选定产物的自上而下蛋白质组学的证实，亚细胞分布的变化将被用作下游封顶对蛋白质多样性的功能影响的分析。综上所述，这项工作将确定细胞质封顶复合体的组织，重排末端在目标转录本中的位置，以及细胞质封顶对转录组和蛋白质组复杂性的影响。

 DESCRIPTION (provided by applicant): This proposal deals with the unprecedented phenomenon of mRNA capping in the cytoplasm. The 5' ends of all mRNAs have an m7G `cap', and proteins that bind to the cap direct the processing, translation and fate of every transcript. The prevailing view was that caps could only be added to newly synthesized pre-mRNAs in the nucleus, and loss of the cap leads irreversibly to mRNA decay. In contrast, we identified transcripts that are stable in an uncapped state, identified a cytoplasmic complex of proteins that can restore the cap onto these transcripts, and identified a cyclical process of decapping and recapping termed `cap homeostasis' that maintains a subset of the transcriptome in an actively translating state. The process of cytoplasmic capping involves conversion of the 5'-monophosphate end of uncapped RNA to a 5'-diphosphate and the transfer of GMP from capping enzyme onto this recapping substrate. All of the enzymes needed to catalyze cytoplasmic capping are present in a single complex that assembles on Nck1, a cytoplasmic SH2/SH3 adapter protein that is best known as a transducer of tyrosine kinase signaling. The RNA 5'-kinase and capping enzyme are juxtaposed by binding to adjacent SH3 domains, and the presence of cap methyltransferase in the complex completes the list of proteins that are necessary and sufficient to affect cytoplasmic capping. Cytoplasmic capping targets are not random; they encode proteins involved in nucleotide binding, protein localization, RNA localization, and the mitotic cell cycle. The working hypothesis of this proposal is that cytoplasmic capping is a selective post-transcriptional process that functions as an amplifier of transcriptome and proteome complexity. In Aim 1 in vitro, in vivo and biochemical biological approaches will be used to characterize the 5'-kinase and its function in cytoplasmic capping. Nck1 has 4 functional domains, and classical and `top-down' proteomics and biochemical approaches will be used to identify and characterize proteins that are bound uniquely to the 1st SH3 domain and SH2 domain in the context of the cytoplasmic capping complex. Aim 2 will map the 5' ends of recapped transcripts and determine their relationship to internal cap sites identified by Capped Analysis of Gene Expression (CAGE). The resulting datasets will be mined to identify sequence and/or structural motifs that determine the location of recapped 5' ends and their role in determining target specificity. In Aim 3 ribosome profiling will be combined with positional proteomics to determine the relationship of cytoplasmic capping to translation and proteome complexity. The results will be confirmed by top-down proteomics of selected products from internally capped transcripts, and changes in subcellular distribution will be used as an assay for functional effects of downstream capping on protein diversity. In summary this work will determine the organization of the cytoplasmic capping complex, the location of recapped ends within target transcripts, and the impact of cytoplasmic capping on transcriptome and proteome complexity.