Analysis of a novel family of organelle-directed RNA binding proteins in    Plasmodium falciparum

CHRISTIAN SCHMITZ-LINNEWEBER (HUB) in partnership with Giel van Dooren (ANU)

Plasmodium cells depend on two (non-nuclear) DNA-bearing organelles, apicoplasts and mitochondria. Gene expression in these organelles is essential for parasite survival, with antibiotics targeting gene expression in the apicoplast and mitochondrion serving as potent antimalarials [1, 2]. Importantly, Plasmodium organellar gene expression depends on a large number of nuclear-encoded proteins that are imported into the organelles post-translationally (e.g. [3]). The details of Plasmodium organellar gene expression, i.e. the individual expression steps as well as the underlying factors, are poorly understood. This includes the enigmatic assembly of the ribosome from the highly fragmented rRNAs in the mitochondrion. The group of Christian Schmitz-Linneweber has recently found short non-coding RNA fragments that are possible footprints of RNA binding proteins (RBPs) in Plasmodium organelles. In plants, such short RNAs are generated by the action of RBPs from the PPR class [4, 5]. Intriguingly, many of the Plasmodium organellar short RNAs (sRNAs) overlap with 5’ ends of rRNA fragments. We hypothesize that these are footprints of RBPs involved in puzzling together the rRNA fragments into a functioning ribosome. A bioinformatics search of the Plasmodium nuclear genome identified a hitherto undescribed family of organellar helical hairpin repeat protein family (that the group term HHR proteins) as well as a limited number of PPR proteins. These proteins are predicted to be imported into the Plasmodium organelles, which we have confirmed for selected candidates. Members of this family are also found within other Apicomplexans, including Toxoplasma gondii, which provides a genetically tractable model for Plasmodium biology. The van Dooren lab has confirmed the presence of selected HHR proteins in T. gondii mitochondria using a proximity labelling approach and fluorescence protein tagging.

This proposal aims at unravelling the function of six HHR proteins for Plasmodium organellar RNA metabolism. (i) The project will confirm their intracellular location in Plasmodium berghei and T. gondii using tagging approaches. (ii) Mutant strains of P. berghei and T. gondii deficient in the expression of candidate proteins will be generated, and the effect on RNA metabolism and parasite survival will be studied. (iii) The RNA ligands of these RBPs will be determined by tested RNA co-immunoprecipitation approaches using either antisera raised against RBP peptides or using Plasmodium lines that are stably expression epitope-tagged RBPs. (iv) The proteomic context of HHRs/PPRs will be analysed to understand their larger network of interactions (as part of the student’s stay at ANU). (v) Suborganellar localization of RNA binding proteins will be determined using high-resolution microscopy.

Interlinkages: Benedikt Beckmann (HUB), Kai Matuschewski (HUB), Melanie Rug (ANU), Frank Seeber (HUB)

References:

(1) Painter, H. J. et al. (2007) Nature 446: 88-91

(2) Afonso, A. et al. (2010) Malaria J. 9: 135

(3) Waller, R. F. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12352-12357

(4) Ruwe, H. et al. (2012) Nucleic Acids Res. 40: 3106-3116

(5) Ruwe, H. et al. (2016) Nucleic Acids Res. in press