Functional Characterization of the m(6)A-Dependent Translational Modulator PfYTH.2 in the Human Malaria Parasite

Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N(6)-methylation of adenosines (m(6)A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m(6)A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m(6)A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m(6)A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.