mRNA decapping by ApaH like phosphatases

• Applicant: Professorin Dr. Susanne Kramer
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Parasitology and Biology of Tropical Infectious Disease Pathogens ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 398051313

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.

• DFG Programme: Research Grants