mRNA decapping by ApaH like phosphatases

ApaH 样磷酸酶对 mRNA 进行脱帽

• 批准号: 398051313
• 负责人: Professorin Dr. Susanne Kramer
• 金额: --
• 依托单位: Lehrstuhl für Zell- und Entwicklungsbiologie (Zoologie I)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/398051313?language=en
• 关键词: mRNA; decapping; ApaH; like; phosphatases

Every eukaryotic mRNA is stabilised at its 5´end by an m7 methylguanosine (m7G) cap. At the end of its life, in the 5´-3´decay pathway, the cap is removed by a decapping enzyme, followed by 5´-3´ exonucleolytic degradation. The prototype of the eukaryotic mRNA decapping enzyme is the nudix domain protein Dcp2. Kinetoplastida lack orthologues to Dcp2 and we recently found that they use an ApaH like phosphatase (Alph) instead. Alph’s originate from the bacterial ApaH protein. Proteins of this family are present in all eukaryotic kingdoms, but apart from the trypanosome Alph, the function of only one further protein is known: the yeast Alph protein cleaves poly(phosphate) in the vacuole.In the first funding period, we have characterised the novel mRNA decapping enzyme of the Kinetoplastida Trypanosoma brucei in vitro. We found broad substrate specificity that even includes cap analogues not attached to RNA and was only dependent on the enzymes catalytic domain, but not on its N- and C-terminal extensions. We searched for Alph proteins throughout the entire eukaryotic kingdom to investigate, whether mRNA decapping by Alphs is a widespread phenomenon: we found that Alphs are either absent or non-cytoplasmic in all eukaryotes except the Kinetoplastida, and, surprisingly all that we tested had mRNA decapping activity. The wide substrate range of Alphs suggests a selective pressure against the possession of cytoplasmic Alphs in eukaryotes to protect the mRNAs from unregulated decapping and degradation. Only Kinetoplastida have succeeded to exploit the mRNA decapping activity of Alph to their advantage, likely by adding unique regulatory domains.The next major step now is to understand, how mRNA decapping activity of Alph is regulated in Kinetoplastida. Towards this aim, we have started to identify Alph interacting proteins and we have started to analyse the phenotypes of various cell lines with truncated or mutated Alph variants. Here, we ask for another 18 month of funding, to finish these in vivo experiments and to understand the mechanism and regulation of ALPH1 in detail. This is only the second characterised Alph protein of any eukaryote and our data will contribute to a better understanding of this enzyme family. Importantly, ALPH1 is a putative drug target against African Sleeping sickness, Leishmaniasis and Chagas disease, as it is essential in trypanosomes but absent in humans. For this reason, we are collaborating with Maria Gorna in Warsaw to solve the structure of ALPH1.

每个真核生物的mRNA在其5 ′端由m7甲基鸟苷（m7 G）帽稳定。在其寿命结束时，在5 ′-3 ′衰变途径中，帽被脱帽酶去除，然后进行5 ′-3 ′核酸外切降解。真核生物mRNA脱帽酶的原型是营养结构域蛋白Dcp 2。动质体缺乏Dcp 2的直系同源物，我们最近发现它们使用ApaH样磷酸酶（Alph）代替。Alphs来源于细菌ApaH蛋白。这个家族的蛋白质存在于所有的真核生物界，但除了锥虫阿尔法，只有一个进一步的蛋白质的功能是已知的：酵母阿尔法蛋白裂解聚（磷酸盐）在vacuole.In第一个资金周期，我们已经确定了新的mRNA脱帽酶的动质体布氏锥虫在体外。我们发现了广泛的底物特异性，甚至包括帽类似物不连接到RNA，只依赖于酶的催化结构域，但不依赖于其N-和C-末端的延伸。我们在整个真核生物界搜索了Alphs蛋白，以研究Alphs对mRNA的去帽是否是一种普遍现象：我们发现除了动质体外，所有真核生物中Alphs要么不存在，要么是非细胞质的，令人惊讶的是，我们测试的所有生物都具有mRNA去帽活性。Alphs的广泛底物范围表明，在真核生物中，对细胞质Alphs的占有存在选择性压力，以保护mRNA免受不受调节的去帽和降解。只有动质体成功地利用了α的mRNA去帽活性，很可能是通过添加独特的调控结构域。现在的下一个主要步骤是了解，α的mRNA去帽活性在动质体中是如何调节的。为了实现这一目标，我们已经开始鉴定α相互作用蛋白，并且我们已经开始分析具有截短或突变的α变体的各种细胞系的表型。在此，我们请求再提供18个月的资金，以完成这些体内实验，并详细了解ALPH 1的机制和调控。这只是任何真核生物的第二个特征化的α蛋白，我们的数据将有助于更好地了解这个酶家族。重要的是，ALPH 1是针对非洲昏睡病、利什曼病和恰加斯病的假定药物靶标，因为它在锥虫中是必需的，但在人类中不存在。出于这个原因，我们正在与华沙的Maria Gorna合作解决ALPH 1的结构。