Life is about balance: Capturing genome-wide mRNA transcription and stabilization during development of the malaria parasite

A simple method for capturing transcriptional activity in the malaria parasite opens the way for dissecting mRNA dynamics during development in human red blood cells.

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Plasmodium falciparum is the most deadly causative agent of the disease malaria and, despite decades of heroic eradication efforts by the global health community, it continues to affect over 200 million people per year. Human infection with Plasmodium parasites is initiated by a bite from an infectious mosquito, followed by development in the liver that results in thousands of parasites being released into the blood.  It is the blood stage of infection that leads to all of the clinical symptoms of malaria including the hallmark cyclical fevers that occur every 2 days as the parasites mature and divide within the blood and burst back out to infect new red blood cells.  Therefore, one of the main symptoms is anemia as the parasite ravages the red blood cell content of the infected host.  To complete the parasite’s lifecycle, mosquitoes take up a differentiated form of the blood stage parasite, a sexual form which will then mature in the mosquito for onward transmission to a new host.  Understanding the regulation of these developmental differentiation processes in the liver, blood and mosquito has been at the core of malaria research for over a century. Despite the completion of the whole genome sequence of P. falciparum over 15 years ago, how the genome is transcribed into mRNA transcripts and the mechanisms of transcriptional regulation throughout the parasite’s complex life cycle remain poorly understood. 

Genome-wide analysis of transcription in the human malaria parasite Plasmodium falciparum has revealed robust variation in steady-state mRNA abundance throughout the 48-hour intraerythrocytic developmental cycle (IDC) suggesting that this process is highly dynamic and tightly regulated.  The steady state amounts of cellular mRNA reflect the rates at which new transcripts are generated and the rate at which they are destroyed.  However, the precise timing of mRNA transcription and decay remains poorly understood due to the utilization of methods that only measure total RNA and cannot differentiate between newly transcribed, decaying and stable cellular RNAs.  In recent years, biosynthetic labeling and capture of nascent mRNA has allowed researchers to determine the dynamics of mRNA transcription and stability within specific cells, in a population of cells over time, and in response to external perturbations.  Given the developmental complexity of Plasmodium parasites, we wondered if biosynthetic RNA labeling could provide us insight into the regulation of mRNA abundance as growth and development occur within the human red blood cells? 

Genome-wide mRNA abundance during Plasmodium asexual development is regulated by various regulatory dynamics

To capture the transcriptional dynamics within proliferating parasites, methods to differentiate nascent transcription from pre-existing mRNAs are necessary.  The approach we chose in our recently published study was to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines.  However, the challenge we faced is that the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis.  Therefore, we had to devise a way to enable these parasites to salvage pyrimidines and incorporate detectable modified pyrimidines.  For this, we engineered parasites to express a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU) tagged with green fluorescent protein (GFP).  Expression of the GFP-tagged FCU was verified by fluorescent imaging and Western blot to ensure the protein was being produced in the parasite.  The product of this gene catalyzes the conversion of cytosine to uracil and uracil to UMP, which can be utilized as a monomer for RNA.  After optimization, we were able to demonstrate that expression of FCU in Plasmodium parasites allowed for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs by biotinylation and Northern blot of ribosomal RNAs after probing with streptavidin-HRP.  Thio-modified RNAs can be selectively isolated from total cellular RNA by a combining biotinylation of the thiol-modification and streptavidin magnetic beads to purify nascent mRNAs.

Confirmation of transgene expression facilitating pyrimidine salvage

Using this exciting new tool we were able to capture and quantify newly-synthesized P. falciparum RNA transcripts at  every hour throughout the 48 hour intraerythrocytic developmental cycle following erythrocyte invasion.  To do this, we pulsed synchronous in vivo cultures of P. falciparum with thiol-modified uracil for 10 min which, ultimately, was incorporated into nascent transcripts.  We then isolated parasite RNA from these cultures and separated the thiolated nascent RNA from total RNA.  Each population of RNA (total, nascent, unlabeled) was converted to complementary DNA (cDNA) by reverse transcription and quantified by cDNA microarray.  The resulting data provide the most high-resolution global analysis of the transcriptome to date and captures both the timing and rate of transcription for each newly synthesized mRNA in vivo as well as the stability of each transcript.  

Schematic representation of the experimental design

With the help of our bioinformatics and modelling collaborators who developed a statistical model for this dataset, we determined the fraction of active transcription and/or transcript stabilization contributing to the total mRNA abundance at each timepoint. Excitingly, this revealed varying degrees of transcription and stabilization for each mRNA corresponding to developmental transitions and independent of abundance profile.  Finally, our results provide new insight into co-regulation of mRNAs throughout the IDC through regulatory DNA sequence motifs, thereby expanding our understanding of P. falciparum mRNA dynamics.

Bioinformatic analysis reveals the contribution of transcription and stabilization to mRNA abundance

This study represents the first time that both transcriptional activity and transcript stabilization has been captured during the IDC in a single experiment.  We envision that these results will provide a useful resource the malaria research community and aid in broadening our understanding of transcriptional regulation.  In addition, the adaptation of biosynthetic labeling and capture of nascent transcription opens new avenues to capture developmental stage specific transcriptional programs by altering the promoter regulating the expression of FCU or measure the parasite’s response to anti-malarial treatment by capturing immediate transcriptional changes.

Manuel Llinás

Professor, The Pennsylvania State University