Relationship of cytoplasmic capping to post-transcriptional gene regulation

 描述（由申请人提供）：该提案涉及细胞质中mRNA加帽的前所未有的现象。所有mRNA的5'末端都有一个m7 G“帽”，与帽结合的蛋白质指导每个转录物的加工、翻译和命运。流行的观点是，帽只能被添加到细胞核中新合成的前mRNA中，帽的丢失会导致mRNA不可逆地衰变。相比之下，我们确定了在未加帽状态下稳定的转录本，确定了可以将帽恢复到这些转录本上的蛋白质的细胞质复合物，并确定了称为“帽稳态”的去帽和重新帽的循环过程，该过程保持转录组的子集处于活跃的翻译状态。细胞质加帽的过程包括将未加帽RNA的5 '-单磷酸末端转化为5'-二磷酸，并将GMP从加帽酶转移到该重新加帽底物上。催化细胞质加帽所需的所有酶都存在于一个单一的复合物中，该复合物组装在Nck 1上，Nck 1是一种细胞质SH 2/SH 3衔接蛋白，最为人所知的是酪氨酸激酶信号转导蛋白。RNA 5 '-激酶和加帽酶通过与相邻的SH 3结构域结合而并列，并且复合物中帽甲基转移酶的存在完成了影响细胞质加帽所必需且足够的蛋白质列表。细胞质加帽靶点不是随机的;它们编码参与核苷酸结合、蛋白定位、RNA定位和有丝分裂细胞周期的蛋白质。这个建议的工作假设是，细胞质加帽是一个选择性的转录后过程，作为一个放大器的转录组和蛋白质组的复杂性。在目标1中，将使用体外、体内和生化生物学方法来表征5 '-激酶及其在细胞质加帽中的功能。Nck 1有4个功能结构域，经典的和“自上而下”的蛋白质组学和生物化学方法将用于鉴定和表征在细胞质加帽复合物的背景下独特地与第一SH 3结构域和SH 2结构域结合的蛋白质。目的2将绘制再加帽转录物的5'端，并确定它们与通过加帽基因表达分析（CAGE）鉴定的内部帽位点的关系。将挖掘所得数据集以鉴定确定重新加帽的5'末端的位置及其在确定靶特异性中的作用的序列和/或结构基序。在目标3中，核糖体分析将与位置蛋白质组学相结合，以确定细胞质加帽与翻译和蛋白质组复杂性的关系。这些结果将通过从内部加帽转录物中选择的产物的自上而下的蛋白质组学来证实，亚细胞分布的变化将被用作下游加帽对蛋白质多样性的功能影响的测定。总之，这项工作将确定组织的细胞质加帽复合物，目标转录内的recapped结束的位置，和细胞质加帽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.