m5C methylation of mitochondrial RNA and non-coding RNA by NSUN3 is associated with variant gene expression and asexual blood-stage development in Plasmodium falciparum.

Background: Malaria is caused by Plasmodium spp. and is a prevalent parasitic disease worldwide. To evade detection by the immune system, by switching variant gene expression, the malaria parasite continually establishes new patterns displaying a single variant erythrocyte surface antigen. The distinct surface molecules encoded by clonally variant gene families include var, rif, stevor, Pfmc-2tm, and surfins. However, the mechanism behind the exclusive expression of a single member of the variant gene family is still not clear. This study aims to describe the molecular process of variant gene switching from the perspective of the epitranscriptome, specifically by characterizing the role of the Plasmodium falciparum RNA m5C methyltransferase NSUN3.

Methods: A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3' untranslated region (UTR) of the pfnsun3 gene. A transgenic parasite line PfNSUN3-Ty1-Ribo was generated using CRISPR-Cas9 methods. The knockdown effect in the transgenic parasite was measured by a growth curve assay and western blot analysis. The transcriptome changes influenced by PfNUSN3 knockdown were detected by RNA sequencing (RNA-seq), and the direct RNA transcripts regulated by PfNUSN3 were validated by RNA immunoprecipitation and high-throughput sequencing (RIP-seq).

Results: Growth curve analysis revealed that conditional knockdown of PfNSUN3 interfered with parasite growth. The parasitemia of the PfNSUN3 knockdown line showed a significant decline at the third round of the life cycle compared with the control line. The knockdown of PfNSUN3 altered the global transcriptome. RNA-seq analysis showed that at the ring-stage depletion of PfNSUN3 silenced almost all var genes, as well as the guanine/cytosine (GC)-rich non-coding RNA (ncRNA) ruf6 family. RNA RIP-seq arrays revealed that PfNSUN3 directly interacted with several var genes.

Conclusions: Our findings demonstrate a vital role of PfNSUN3 in the process of the mutually exclusive expression of variant genes, and contribute to a better understanding of the complex mechanism of epigenetic regulation of gene expression in P. falciparum.