Parasite Genetics and Adaption - Project B1

RNA Processing and turnover in Apicomplexan organelles

Host labs: C. Schmitz-Linneweber, Molecular Genetics at Humboldt University Berlin; Melanie Rug, Alex Maier, ANU Canberra

Partner labs: Kai Matuschewski, HU Berlin: P. berghei genetics; Frank Seeber, RKI Berlin: T. gondii genetics, Melanie Rug, ANU Canberra:                             cryoelectron tomography

Background: Apicoplasts and mitochondria contain their own genetic information. Gene expression in these organelles is essential for parasite survival. Antibiotics targeting gene expression in the apicoplast and mitochondrion serve as potent antimalarials. Very little is known about the organellar gene expression machinery in Apicomplexans. We recently identified a family of RNA binding proteins named heptatricopeptide repeat proteins (HPR proteins; Hillebrand et al., 2018). Members of this family were demonstrated to reside in RNA granules in human mitochondria (Jourdain et al., 2017) and play a role in rRNA and mRNA processing. Initial gene tagging and knock-out approaches demonstrated that these proteins reside in mitochondria and are essential for parasite survival. Their exact functions in mitochondrial RNA metabolism remains enigmatic. In general, RNA turnover and its regulation is hardly addressed in Apicomplexans, neither in organelles nor in the nucleo-cytoplasmic compartment. This project aims at filling this gap in our knowledge.

Central Question: What is the role of Apicomplexan HPR proteins in RNA processing and stabilization?

Methods employed:

• Generation of mutants and tagged lines of HPR genes in Toxoplasma gondii
• Measuring RNA turnover in HPR mutants by metabolic labeling and RNA Next Generation Sequencing (ERIC-Seq)
• Analysis of mitochondrial RNA processing in HPR mutants by Nanopore Sequencing
• CLIP-Seq of HPR proteins to identify their RNA binding sites
• Analysis of HPR localization within mitochondrial RNA granules using STED microscopy and cryoelectron tomography
• Identification of proteins interacting with HPR proteins using proximity labelling

Outline of 3-year plan:

Year 1: Berlin. Preparation of tagged lines, knock-out mutants, and inducible mutants of HPR proteins. Mutant analysis using RNA Seq,                     ERIC-Seq and Nanopore sequencing.

Year 2: Canberra. Search for interaction partners of HPR proteins using proximity labelling. Analysis of the localization of HPR proteins                      using cryo-lectron tomography

Year 3: Berlin. CLIP-Seq of selected HPR proteins. STED microscopy of HPR proteins and RNA granules. Writing up thesis.

Keywords: Mitochondria, RNA processing and turnover, HPR proteins

Reference: Hillebrand, A., Matz, J.M., Almendinger, M., Muller, K., Matuschewski, K., and Schmitz-Linneweber, C. (2018). Identification of clustered organellar short (cos) RNAs and of a conserved family of organellar RNA-binding proteins, the heptatricopeptide repeat proteins, in the malaria parasite. Nucleic Acids Res. 46(19):10417-10431. doi: 10.1093/nar/gky710.