Deciphering the Plasmodium epitranscriptome as a mechanism of translational control

Abstract

Modifications to nucleosides present in ribonucleic acid (RNA) species have been shown to influence gene expression. Using a LC-MS/MS-based platform, we have characterized the full spectrum of modified ribonucleosides on the transfer RNA (tRNA) and messenger RNA (mRNA) in P. falciparum. We demonstrate that modifications on the wobble position of certain tRNA isoacceptors are associated with efficient translation of corresponding codon biased genes. Of the mRNA modifications, we quantified N6-methyladenosine (m6A) across the intra- erythrocytic lifecycle and characterized the putative “writer” enzyme complex that catalyses this modification. Upon carrying out m6A-sequencing, we find that m6A-associated transcripts have shorter half-lives and lower translation efficiency. Additionally, we quantified tRNA modification levels in the parasites that were exposed to oxidative stressors like hydrogen peroxide, sodium hypochlorite and anti-malarials like artemisinin and chloroquine. The work presented here holds potential for several avenues of new discovery. For one, unique patterns of tRNA modification reprograming can distinguish mechanistically distinct damaging agents which supports the idea the tRNA modification patterns can be used as biomarkers of exposure. Besides these stress-specific patterns of tRNA modification changes are linked to selective translation of codon-biased mRNAs for stress response proteins in other organisms. In order to address this hypothesis in Plasmodium we have carried out a proteomics experiment to determine if the parasite proteins that are upregulated show a marked codon bias which can be tied up with the tRNA modification data. All of these findings together provide new insights into the role of translational control mechanisms involving RNA modifications in the development and pathogenesis of malaria parasites.