mRNA decapping by ApaH like phosphatases

每个真核mRNA在其5′端被m7甲基鸟苷（m7G）帽稳定。在其生命结束时，在5′-3′衰变途径中，帽被脱帽酶去除，随后进行5′-3′外核溶解降解。真核生物mRNA脱帽酶的原型是裸结构域蛋白Dcp2。动质体缺乏与Dcp2的同源物，我们最近发现它们使用类似ApaH的磷酸酶（Alph）代替。Alph来源于细菌的ApaH蛋白。这个家族的蛋白质存在于所有真核生物王国中，但除了锥虫的Alph，只有一种蛋白质的功能是已知的：酵母的Alph蛋白在液泡中切割聚磷酸盐。在第一个资助期，我们在体外鉴定了布鲁氏动胞体锥虫的新型mRNA脱帽酶。我们发现了广泛的底物特异性，甚至包括不附着在RNA上的cap类似物，并且仅依赖于酶的催化结构域，而不依赖于其N端和c端延伸。我们在整个真核生物王国中搜索了Alph蛋白，以研究Alphs对mRNA脱帽是否普遍存在：我们发现除了动质体外，所有真核生物中都不存在Alphs或非细胞质，并且令人惊讶的是，我们测试的所有真核生物都具有mRNA脱帽活性。Alphs的广泛底物范围表明，真核生物对细胞质Alphs的占有存在选择性压力，以保护mrna免受不受管制的脱帽和降解。只有着丝质体成功地利用了Alph的mRNA脱帽活性，可能是通过添加独特的调控结构域。下一个主要步骤是了解在着丝质体中Alph的mRNA脱帽活性是如何被调节的。为了实现这一目标，我们已经开始鉴定Alph相互作用蛋白，并开始分析各种截断或突变的Alph变异细胞系的表型。在此，我们要求再提供18个月的资金，以完成这些体内实验，并详细了解ALPH1的机制和调控。这是在真核生物中唯一鉴定出的第二个Alph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，ALPH1是针对非洲昏睡病、利什曼病和恰加斯病的假定药物靶点，因为它在锥虫中必不可少，但在人类中不存在。因此，我们正在与华沙的Maria Gorna合作解决ALPH1的结构。

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.