Periodic Reporting for period 2 - PlasmoEpiRNA (Resolving m6A-mediated post-transcriptional control in the human malaria parasite)
Berichtszeitraum: 2022-12-01 bis 2024-05-31
The human malaria parasite, Plasmodium falciparum, successfully replicates within and transmit between multiple different tissues and organisms, each representing a new environment the parasite rapidly adapts to. In preparation for the sudden switch from human host to mosquito vector, the parasite develops into a transient quiescent transmission stage, gametocytes. One regulatory mechanism of gametocytes is the selective translation of some and translational repression of other mRNAs. Since translationally repressed transcripts mostly include those needed after transmission, the parasite transcriptome is ‘primed’ for the new host environment and a more rapid re-initiation of development than might be achievable by adapting transcriptional or epigenetic gene regulation to the new environment. However, a mechanism or mRNA feature or specific RNA-binding proteins that can mediate mRNA triage for translation or repression, sequestration into subcellular compartments, and, most importantly, de-repression after transmission remain largely elusive. We had previously identified the methylation of adenosines (m6A) at internal mRNA positions as an additional layer of post-transcriptional regulation in the malaria parasite. Given its dynamics throughout the parasite life cycle, we hypothesize that m6A is the missing link of post-transcriptional regulation in parasite transmission stages.
The overall goal of this proposal is to 1) investigate how m6A mediates translational repression and mRNA stability during proliferation in the human through the selective binding of m6A reader proteins and 2) elucidate its role in mediating transient quiescence and ‘priming’ of the transcriptome in preparation for the environmental changes accompanying transmission between host and vector.
The extent and dynamics of m6A in P. falciparum make it stand out among characterized methylation programs in eukaryotic model organisms, none of which undergo similar changes in environment as this pathogen during transmission from host to vector or vice versa. Given the complexity of the Plasmodium life cycle, we anticipate that this pathogen may have evolved a unique m6A methylation program to post-transcriptionally regulate gene expression in a wide range of environments. Importantly, the unique sequence of proliferative and non-proliferative stages will allow us to gain detailed mechanistic insight into regulatory mechanisms underlying possibly m6A-mediated transient quiescence and the exit thereof.
Transmission stages represent one of the most crucial bottlenecks in the lifecycle of the malaria parasite. Understanding the processes that allow the parasite to successfully pass through them could ultimately also lead to new approaches to combat the spread of the disease.
The overall goal of this proposal is to 1) investigate how m6A mediates translational repression and mRNA stability during proliferation in the human through the selective binding of m6A reader proteins and 2) elucidate its role in mediating transient quiescence and ‘priming’ of the transcriptome in preparation for the environmental changes accompanying transmission between host and vector.
The extent and dynamics of m6A in P. falciparum make it stand out among characterized methylation programs in eukaryotic model organisms, none of which undergo similar changes in environment as this pathogen during transmission from host to vector or vice versa. Given the complexity of the Plasmodium life cycle, we anticipate that this pathogen may have evolved a unique m6A methylation program to post-transcriptionally regulate gene expression in a wide range of environments. Importantly, the unique sequence of proliferative and non-proliferative stages will allow us to gain detailed mechanistic insight into regulatory mechanisms underlying possibly m6A-mediated transient quiescence and the exit thereof.
Transmission stages represent one of the most crucial bottlenecks in the lifecycle of the malaria parasite. Understanding the processes that allow the parasite to successfully pass through them could ultimately also lead to new approaches to combat the spread of the disease.
During the reporting period, we have identified and characterized the functions of two proteins that specifically bind m6A-methylated mRNA during asexual replication in the human malaria parasite. We developed and adapted methods to measure the extent and dynamics of m6A at single nucleotide resolution during gametocyte development. By inducible disruption of the m6A methylation program we were able to show how it affects the development of the transmission stages and its effect on mRNA translation. We further adapted methods to identify protein-mRNA interactions at single-nucleotide resolution across the transmission stage development and by combining multiple different microscopy approaches, we are now able to follow the subcellular dynamics of m6A ‘reader’ proteins and their cognate m6A-methylated mRNA in vivo.
For the first time, we are able to identify a mRNA ‘feature’ (i.e. m6A) as mediator of a specific RNA-protein interaction in the malaria parasite. Tracing the effect of this specific interaction on a molecular and cellular level, we could reveal how it affects parasite development on a organismal level. By combining several novel genomic and microscopy approaches for the malaria parasite, we are now able in a next step to reveal the effect of sudden environmental changes on mRNA-protein interactions and characterize how this regulates parasite development during transmission.