Abstract

BackgroundPlasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood. The N6-methyl adenosine (m6A) is an important epigenetic modification primarily present on mRNA that controls the levels of transcripts and efficiency of translation in eukaryotes. Recently, the dynamics of m6A on mRNAs at all three developmental stages of P. falciparum in RBCs have been profiled; however, the proteins that regulate the m6A containing mRNAs in the parasites are unknown.ResultsUsing sequence analysis, we computationally identified that the P. falciparum genome encodes two putative YTH (YT521-B Homology) domain-containing proteins, which could potentially bind to m6A containing mRNA. We developed a modified methylated RNA immunoprecipitation (MeRIP) assay using PfYTH2 and find that it binds selectively to m6A containing transcripts. The PfYTH2 has a conserved aromatic amino acid cage that forms the methyl-binding pocket. Through site-directed mutagenesis experiments and molecular dynamics simulations, we show that F98 residue is important for m6A binding on mRNA. Fluorescence depolarization assay confirmed that PfYTH2 binds to methylated RNA oligos with high affinity. Further, MeRIP sequencing data revealed that PfYTH2 has more permissive sequence specificity on target m6A containing mRNA than other known eukaryotic YTH proteins. Taken together, here we identify and characterize PfYTH2 as the major protein that could regulate m6A containing transcripts in P. falciparum.ConclusionPlasmodium spp. lost the canonical m6A-specific demethylases in their genomes, however, the YTH domain-containing proteins seem to be retained. This study presents a possibility that the YTH proteins are involved in post-transcriptional control in P. falciparum, and might orchestrate the translation of mRNA in various developmental stages of P. falciparum. This is perhaps the first characterization of the methyl-reading function of YTH protein in any parasites.

Highlights

  • Plasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood

  • We found that PfYTH2 protein contains conserved aromatic amino acids that are essential to bind methylated adenosine (Additional file 1: Figure S1)

  • We found strong binding of PfYTH2 protein to m6A-containing RNAs compared to negligible binding in the GST control samples (Fig. 1b)

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Summary

Introduction

Plasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood. Developmental switches in P. falciparum are marked by broad changes in transcriptional activity [4,5,6]. These findings suggest that Plasmodium spp. have distinct regulatory mechanisms, both at the post-transcriptional and translational levels that regulate various developmental stages of the Plasmodium lifecycle. Such unique mechanisms to fine-tune the global gene expression might help P. falciparum to establish successful pathogenesis inside the human host

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