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Charting new territory: The Plasmodium falciparum tRNA modification landscape


This review addresses the known functions of tRNA modifications in the biology ofP. falciparumwhile highlighting the potential therapeutic opportunities and the value of usingP. falciparumas a model organism for addressing several open questions related to the tRNA epitranscriptome.



Abstract

Ribonucleoside modifications comprising the epitranscriptome are present in all organisms and all forms of RNA, including mRNA, rRNA and tRNA, the three major RNA components of the translational machinery. Of these, tRNA is the most heavily modified and the tRNA epitranscriptome has the greatest diversity of modifications. In addition to their roles in tRNA biogenesis, quality control, structure, cleavage, and codon recognition, tRNA modifications have been shown to regulate gene expression post-transcriptionally in prokaryotes and eukaryotes, including humans. However, studies investigating the impact of tRNA modifications on gene expression in the malaria parasite Plasmodium falciparum are currently scarce. Current evidence shows that the parasite has a limited capacity for transcriptional control, which points to a heavier reliance on strategies for posttranscriptional regulation such as tRNA epitranscriptome reprogramming. This review addresses the known functions of tRNA modifications in the biology of P. falciparum while highlighting the potential therapeutic opportunities and the value of using P. falciparum as a model organism for addressing several open questions related to the tRNA epitranscriptome.


Introduction

Gene expression is a highly coordinated process that is regulated at multiple levels in every organism. At the post-transcriptional level, gene expression is orchestrated through several mechanisms that ultimately determine transcript abundance, localization and, in the case of protein-coding genes, the efficiency of translation of the respective transcripts. These mechanisms often involve RNA-RNA and RNA-protein interactions, and the dozens of ribonucleoside modifications that form the epitranscriptome.

Ribonucleoside modifications are known to influence gene expression both locally and in a global, systemic manner. These chemical modifications can occur on either the base or sugar moieties of the nucleotide residues and over 150 of them have so far been identified across all three domains of life1. The presence of these modifications on the three types of RNA directly involved in the ribosomal synthesis of proteins hints at their importance in maintaining tight control over this essential process.

On mRNA, ribonucleoside modifications are scarce but have important roles in influencing transcript stability, localization, and interactions with RNA-binding proteins, including those responsible for the initiation of translation (reviewed in ref 2). Only a handful of mRNA modifications have been identified at present. These modifications can be found throughout the mRNA transcript, with some enriched in the coding sequences while others are more likely to be found in the untranslated regions. The effect that these modifications have on gene expression tend to be local, affecting only the genes of the transcripts that they modify.

rRNA modifications, on the other hand, tend to act more globally, affecting large subsets of transcripts. rRNA typically have a higher abundance of ribonucleoside modifications in comparison to mRNA, and these rRNA modifications appear to be necessary for proper biogenesis and assembly of ribosomes. They tend to be enriched at positions near functional centers of the ribosome, such as the peptidyl transferase and decoding centers3, suggesting functional roles for these modifications.

tRNA modifications can have both local and global effects on genetic regulation. Modifications on specific isoacceptors alter the expression of entire subsets of transcripts based on codon composition. Having the greatest diversity of modifications in terms of chemical identity and function, they also harbor many modifications with highly complex side chains. While modifications are found throughout the tRNA molecule, most of these complex modifications are exclusively on the anticodon stem loop (ASL). Each tRNA molecule is heavily modified with eukaryotic organisms having an average of 13 for their cytoplasmic tRNAs and 5 for their mitochondrial ones (reviewed in ref 4). Over the last two decades, our understanding of the functions of these modifications on tRNA have greatly expanded.

Recent technological advances in mass spectrometry5, 6, next-generation sequencing (NGS)7, 8, 9 and nanopore sequencing10 have led to rapid advances in understanding the function of tRNA modifications and their relevance to health and disease. Much of this knowledge is derived from studies performed on well-studied organisms such as Escherichia coli11, 12, Saccharomyces cerevisiae5, 13, 14, 15, Drosophila melanogaster16, mice17, 18, and humans19, 20, 21, as well as in human diseases such as cancer22, 23, 24, 25. In the apicomplexan parasite Plasmodium falciparum, which is the causative agent in most of the severe cases of malaria in humans, tRNA epitranscriptomics is still largely unexplored. However, techniques like RNA bisulfite sequencing, ribonucleoside mass spectrometry and mass spectrometry of oligonucleotides obtained from RNase T1 digestion of tRNA have provided some initial insights into the P. falciparum tRNA modification landscape26, 27. Given the observation that the parasite has limited ability to control gene expression at the transcriptional level28, 29, a deeper inquiry into the tRNA epitranscriptome could foster a better understanding of genetic regulation in P. falciparum.

In this review, the functions of the tRNA modifications that have been identified so far in P. falciparum will be examined. While in most cases, functional characterization of these modifications has not yet been performed in P. falciparum, their functions can nonetheless be inferred from observations made in other organisms. Also discussed will be the therapeutic and scientific opportunities that can arise from the foray into this nascent field.


Molecular functions of tRNA modifications

Controlling tRNA aminoacylation

tRNAs are initially transcribed as precursor tRNAs (pre-tRNAs) by RNA polymerase III (Pol III). These pre-tRNAs contain intronic, 5’ leader, and 3’ trailer sequences that must be removed as part of its maturation process. The mature tRNA molecule must then be aminoacylated with its cognate amino acid before it can be used for protein translation by the ribosome. The deposition of ribonucleoside modifications on tRNA begins during the maturation process, as evidenced by the presence of modifications at ten positions of pre-tRNA-Phe in S. cerevisiae prior to the removal of its intron by splicing machinery30. It is unclear whether any of these modifications are required for its maturation and there are currently no known examples of modified ribonucleosides being necessary for the processing of pre-tRNA into mature tRNAs.

Aminoacylation of mature tRNAs, however, is a process that sometimes requires specific tRNA modifications to be present. The first known example of this was found in the prevention of mischarging of E. coli tRNA-Ile-CAU with methionine by the modification of the C34 nucleotide to lysidine11. Also in E. coli tRNA, the glutamyl-tRNA synthetase has a 520-fold higher specificity for tRNA-Glu that contains the mnm5s2U34 modification12. In S. cerevisiae tRNA, m1G37 on tRNA-Asp-GUC prevents its mischarging with arginine13 and modification of A34 of tRNA-Ile-AAU to inosine (I) increases its aminoacylation with isoleucine by more than 15-fold14.

While analogous mechanisms for influencing the rate and specificity of aminoacylation of tRNA have not been documented in P. falciparum, some of the modifications discussed here, like m1G and I, are also present in P. falciparum tRNA27. While it is possible that these modifications are involved in processes relating to tRNA aminoacylation in P. falciparum, it is also feasible that they perform functions other than, or in addition to, controlling aminoacylation.


Facilitating tRNA transport

Ribonucleoside modifications also influence the transport of tRNA between cellular compartments. Recombinant human exportin-t, which exports tRNA from the nucleus, has a binding affinity for mature tRNA 10-times stronger than in vitro transcribed tRNA lacking post-transcriptional modifications. This observation implies that tRNA modifications may participate in tRNA export to the cytosol (Figure 1a), possibly by enhancing the binding of exportin-t to its targets31.


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