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m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii

Michael J. Holmes, Leah R. Padgett, Matheus S. Bastos, William J. Sullivan Jr.

Posted on: 22 March 2021

Preprint posted on 29 January 2021

Article now published in PLOS Pathogens at http://dx.doi.org/10.1371/journal.ppat.1009335

and

A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental genes partitioning in Toxoplasma

Dayana C. Farhat, Matthew Bowler, Guillaume Communie, Dominique Pontier, Lucid Belmudes, Caroline Mas, Charlotte Corrao, Yohann Couté, Alexandre Bougdour, Thierry Lagrange, Mohamed-Ali Hakimi, Christopher Swale

Posted on:

Preprint posted on 23 February 2021

Article now published in eLife at http://dx.doi.org/10.7554/eLife.68312

The never-ending transcripts – how m6A RNA modifications regulate mRNA transcription termination in Toxoplasma gondii

Selected by Joao Mello-Vieira

Categories: cell biology, microbiology

Background

Toxoplasma gondii are eukaryotic parasites that can infect any warm-blooded animals, including humans. In fact, it is thought that one third of the human population is infected with Toxoplasma parasites. In most cases the infection is benign but it can cause serious health hazards to immune compromised individuals and can cause congenital birth defects. Currently there are few therapeutic options to treat toxoplasmosis. In these two preprints, Holmes, et al., and Farhat, et al., discover an important pathway for messenger RNA (mRNA) processing and stability in Toxoplasma gondii parasites, that might serve as a new therapeutic target for anti-Toxoplasma interventions.

This parasite possesses a complex life cycle inside mammals. Humans are infected by either ingestion of infected meat or from contact with infected felines. As the infection progresses parasites differentiate into cysts (namely in the brain) or bradyzoites. These forms are quiescent and cannot be targeted by current therapies.

Transitions between cellular stages, such as in differentiation, are increasingly associated with regulation at the mRNA level, in addition to transcriptional changes. As is the case with DNA, mRNA can also be modified, that affect its stability, localization and translation. mRNA modifications, or the epitranscriptome, seems to be present in eukaryotic kingdoms and its role in cellular processes is still being unravelled. The most common modification is the methylation of adenosine residues, termed m6A. Nonetheless, some proteins have been identified as m6A writers, readers or erasers, depending on the function they perform on the epitranscriptome.

These two preprints look at m6A methylation in Toxoplasma parasites from different approaches. On one hand, Holmes et al. aimed to understand how this modification might help the transition between the active and the quiescent forms of the Toxoplasma parasite. These authors focused mostly on the m6A writer protein METTL3. On the other hand, Farhat, et al., focused on the reader proteins, namely the CPSF complex, that seems to have functions that are shared between Toxoplasma and the plant Arabidopsis thaliana.

 

Key findings from Holmes et al.

  • Toxoplasma parasites have a set of transcripts that possess m6A modifications. These transcripts have adenosine methylation, on the central adenosine of the consensus sequence YGCAUGCR (Y represents any pyrimidine and R any purine), that is more abundant in the 3’ untranslated regions of transcripts;
  • Sequence homology suggested that the Toxoplasma protein METTL3 might be an m6A writer. Indeed, knock-down of METTL3 reduces m6A levels in these transcripts
  • Lack of m6A modification led to the premature degradation of these mRNAs. Importantly, knock-down of METTL3 lead to the arrest of parasite proliferation;
  • Finally, the Toxoplasma gondii YTH m6A reader proteins associate with the cleavage and polyadenylation machinery that catalyses the 3’-end processing of pre-mRNAs.

 

Key findings from Farhat et al.

  • Using biochemical and structural approaches, the authors showed that one of the main subunits of the cleavage and polyadenylation complex CPSF4 has an additional putative YTH domain (m6A reader) giving it the ability to bind to m6A-modifed RNA.
  • Surprisingly, knock-down of the Toxoplasma protein CPSF4 led to the accumulation of very long transcripts in some loci. These transcripts extend beyond the annotated 3’ ends of the gene, ignoring the polyadenylation signal, forming mRNA readthroughs into the adjacent gene. Knock-down of the m6A writer METTL3 caused the same phenotype in these loci.
  • Some of the m6A sites can be tracked by direct RNA sequencing (Oxford Nanopore) using differential error detection between wild type and METTL3 knock-down conditions. The consensus sequence detected ARAC (R=purines) is similar to what has already been observed in Arabidopsis thaliana and other organisms.
  • This dependence on m6A modification to terminate mRNA transcription is similar to that of the model plant Arabidopsis thaliana; when its own m6A writer or reader proteins are mutated, over-extended transcripts appear.
  • Importantly, not all genes need m6A modifications for transcription termination and poly-adenylation. However, it seems that transcripts which termination is m6A-driven often precede stage-specific repressed genes.

 

Significance

Both studies provide mechanistic insight into mRNA transcription termination in Toxoplasma parasites. m6A modification of the 3’ region of some transcripts regulates its transcription termination and is essential for polyadenylation. If this modification is absent, the mRNAs fail to terminate at the appropriate location and PolII transcription can pursue as an aberrant readthrough within the following genes. This affects not only the stability of this new chimeric mRNA but it might also affect downstream protein expression.

Yet, not all genes depend on m6A modifications for the termination of transcription. The lack of m6A seems to be more important for tachyzoite-specific genes leading into stage specifically repressed loci. For example, if the m6A modification is missing, tachyzoite gene transcripts are extended into the next gene, which then terminates at a secondary polyadenylation site. There is probably a link between m6A writer activity on gene loci with downstream stage specific genes in a poised chromatin state.

Interestingly, there seems to be no conventional m6A erasers in the Toxoplasma genome, at least, none that are conserved with other eukaryotes. This might suggest that the m6A mark is irreversible. Additionally, due to sequential and structural divergence, the targeting of either m6A writer or reader complexes could prove to be a viable therapeutic strategy against these parasites.

Parasitic organisms are unique models to study molecular mechanisms, not only because of their clinical importance but also because of their resourcefulness and adaptability. Here, two preprints show how a simple RNA modification, m6A controls the proper termination of transcription of these mRNAs. This mechanism may contribute to the fine tuning of gene expression during different stages. This is critical as the same genome has to be functional in different environments such across different tissues and across different animals. The study of these proteins and their regulators will shed more light on the role of m6A modification, not only in Toxoplasma gondii, but across all kingdoms of life.

Tags: epitranscriptome, m6a, oxford nanopore, polyadenylation, toxoplasma gondii

doi: https://doi.org/10.1242/prelights.27802

(1 votes)

Author's response

William Sullivan Jr shared about m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii

  • Do you think that m6A writer/reader proteins are the same between the different stages of the parasite life cycle? Or do you think that there is a tachyzoite-specific strategy (METLL3 and CSPF proteins) and another bradyzoite-specific mechanism?

William Sullivan Jr (WS): Good question – the answer is unknown. If there are different writers and readers operating in Toxoplasma, they are likely to be novel because the genome does not contain clear additional homologues to these proteins. We suspect if there are stage-specific differences, these would lie in the regulatory proteins associating with the complex. We found a number of hypothetical proteins of unknown function associated with the complexes that are attractive candidates for regulatory roles.

 

  • Do you think that by affecting the m6A transcript termination mechanism, you might also target the quiescent stages of the parasites? Or is this strategy limited to the more metabolically active tachyzoite forms?

WS: I think it depends on the state of the bradyzoite. We are now learning that bradyzoites within cysts are more heterogenous than previously believed, meaning some may still be dividing. I suspect dividing bradyzoites would be more affected by disruptions to m6A. But we and others have shown in other studies that some drugs can reduce brain cyst counts in mice, suggesting that therapeutic elimination of latent bradyzoite cysts is possible. Time will tell if the m6A system is critical to maintain mature bradyzoites. I also think the reactivation process (bradyzoite reconverting back into a tachyzoite) would be adversely affected, which could be clinically relevant. There’s no question that targeting m6A should be detrimental to tachyzoites.

 

  • How easy would it be to target these proteins? In other words, how similar are the Toxoplasma proteins (METTL3/METTL14 and the proteins from the CPSF complex) to the human counterparts?

WS: The core writer components are conserved, but that hasn’t stopped pharmacologists before. In fact, the current frontline treatment for toxoplasmosis inhibits DHFR, an enzyme in the folate pathway that is conserved in humans. The key is there must be structural differences that can be exploited, whereby small molecules bind to the parasite enzyme more tightly than the human counterpart. To avoid the chance of inhibiting the human counterpart too much, a better approach could be to target the proteins we found in association with METTL3 that are unique to the parasite (assuming they are essential for parasite viability). The same principles would apply to the CPSF complex.

 

  • You show an unexpected link between the m6A dependency of Toxoplasma and Arabidopsis. How much can Apicomplexan biology benefit from molecular mechanisms of plants? Is this shared ancestry a particularity of Apicomplexans or is it prevalent in all Protozoa?

WS: The ancestors of the Apicomplexa were around before plants and animals diverged, so there are many plant-like features retained in this group of parasites. There’s also a lineage of transcription factors called AP2 factors that are plant-like.

and

Mohamed-Ali Hakimi and Christopher Swale shared about A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental genes partitioning in Toxoplasma

  • Do you think that m6A writer/reader proteins are the same between the different stages of the parasite life cycle? Or do you think that there is a tachyzoite-specific strategy (METLL3 and CSPF proteins) and another bradyzoite-specific mechanism?

Mohamed-Ali Hakimi and Christopher Swale (MAH/CS): There is no evidence to suggest that the enzymes would be different. Though their enzymatic activity and abundance may be differentially regulated in these stages.

 

  • Do you think that by affecting the m6A transcript termination mechanism, you might also target the quiescent stages of the parasites? Or is this strategy limited to the more metabolically active tachyzoite forms?

MAH/CS: Good question. I think it could work on any stage as mRNA processing is one of the most indispensable activities in the cell, though the sensitivity is probably stronger within the tachyzoite for the very reason you mentioned.

 

  • How easy would it be to target these proteins? In other words, how similar are the Toxoplasma proteins (METTL3/METTL14 and the proteins from the CPSF complex) to the human counterparts?

MAH/CS: Well, we already discovered some inhibitors targeting the parasitic CPSF3 which shares a strong structural conservation between parasites and humans (Bellini et al., 2020 and Swale et al., 2019). The same approach can probably work as well on METTL3/METTL14 as these enzymes are highly essential and may have sufficient divergence to design specific inhibitors.

 

  • You show an unexpected link between the m6A dependency of Toxoplasma and Arabidopsis. How much can Apicomplexan biology benefit from molecular mechanisms of plants? Is this shared ancestry a particularity of Apicomplexans or is it prevalent in all Protozoa?

MAH/CS: It is one of the most fascinating aspects of Apicomplexan parasites (which include Toxoplasma). They have many proteins and biological pathways inherited from red algae through an endosymbiosis that appeared earlier in evolution. Together with machineries belonging to higher eukaryotes, these organisms display a remarkable set of genes.

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