Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development

Baumgarten, Sebastian; Bryant, Jessica M.; Sinha, Ameya; Reyser, Thibaud; Preiser, Peter R.; Dedon, Peter C.; Scherf, Artur

doi:10.1038/s41564-019-0521-7

Cited by 75 publications

(127 citation statements)

References 82 publications

(210 reference statements)

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“…Although AP2 TFs have been at the center of gene expression studies in Plasmodium, novel "omics" approaches have begun uncovering other layers of gene regulation. Indeed, posttranscriptional regulation, such as N 6 -methyladenosine (m 6 A) of mRNA and alternative splicing, have recently been recognized as essential for finetuning gene expression in blood and sexual stages (26,27). In particular, disruption of the splicing factor PbSR-MG was shown to perturb sex-specific alternative splicing, thus demonstrating its role as a cellular differentiation regulator (28).…”

Section: Discussionmentioning

confidence: 99%

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

Toro-Moreno

Sylvester

Srivastava

et al. 2020

mBio

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The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive transcriptome sequencing (RNA-Seq) analysis throughout the Plasmodium berghei LS, covering as early as 2 h postinfection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with coexpression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasite’s LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCE The LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasite’s biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provide insights into these early parasite fluxes that may facilitate establishment of infection, transformation, and replication in the liver.

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Section: Discussionmentioning

confidence: 99%

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

Toro-Moreno

Sylvester

Srivastava

et al. 2020

mBio

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“…While AP2 TFs have been at the center of gene expression studies in Plasmodium , novel “omics” approaches have begun uncovering other layers of gene regulation. Indeed, post-transcriptional regulation, such as N 6 -methyladenosine (m 6 A) of mRNA and alternative splicing, have recently been recognized as essential for fine-tuning gene expression in blood and sexual stages (27, 28). In particular, disruption of the splicing factor Pb SR-MG was shown to perturb sexspecific alternative splicing, thus demonstrating its role as a cellular differentiation regulator (29).…”

Section: Discussionmentioning

confidence: 99%

RNA-Seq Analysis Illuminates the Early Stages ofPlasmodiumLiver Infection

Toro-Moreno¹,

Sylvester

Srivastava³

et al. 2019

Preprint

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24The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease 25 that poses a significant global health burden. Plasmodium spp. initiate infection of the human host 26 by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood 27 when compared to other Plasmodium life stages, which has hindered our ability to target these 28 parasites for disease prevention. We conducted an extensive RNA-seq analysis throughout the 29 Plasmodium berghei LS, covering as early as 2 hours post infection (hpi) and extending to 48 hpi. 30Our data revealed that hundreds of genes are differentially expressed at 2 hpi, and that multiple 31 genes shown to be important for later infection are upregulated as early as 12 hpi. Using 32 hierarchical clustering along with co-expression analysis, we identified clusters functionally 33 enriched for important liver-stage processes such as interactions with the host cell and redox 34 homeostasis. Furthermore, some of these clusters were highly correlated to the expression of 35ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding 36 motifs. This finding presents potential LS targets for these transcription factors, while also hinting 37 at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our 38 work presents a window into the previously undescribed transcriptome of Plasmodium upon host 39 hepatocyte infection to enable a comprehensive view of the parasite's LS. These findings also 40 provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. 41 berghei to human-infective Plasmodium parasites. 42 43 IMPORTANCE 44The LS of Plasmodium infection is an asymptomatic yet necessary stage for producing 45 blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle 46 can prevent clinical malaria, but relatively less is known about the parasite's biology at this stage. 47 Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as 48 early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) 49have not been accessible before due to the technical challenges associated with liver-stage 50 infections. Our data now provides insights into these early parasite fluxes that may facilitate 51 establishment of infection, transformation and replication in the liver. 52 INTRODUCTION 54Plasmodium spp., the causative agents of malaria, are eukaryotic parasites with a largely 55 conserved and complex life cycle that begins in the mammalian host by invasion of hepatocytes. 56In these host cells, a single parasite, termed a sporozoite, will transform and then replicate 57 asexually to form thousands of merozoites, or blood-infective forms (1). After maturation and 58 release from the liver, parasites replicate within erythrocytes causing the clinical manifestation of 59 malaria. Some parasites differentiate into sexual forms ...

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“…MspJI is a restriction endonuclease that cleaves specific motifs containing methylated cytosines that has been used to selectively digest human DNA prior to parasite DNA sequencing [11]. The success of this approach is based on the assumption of different methylation patterns in the human and parasite genomes; however, methylation patterns in P. falciparum are not fully understood [12]. sWGA of the parasite genome over the human genome has also shown promising results as a method for enrichment of parasite DNA prior to whole genome sequencing.…”

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confidence: 99%

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples

Shah

Adams

Moser

et al. 2020

Malar J

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Background: Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined.Methods: DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA.Results: MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI + sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. Conclusion:This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article'

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Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development

Cited by 75 publications

References 82 publications

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

RNA-Seq Analysis Illuminates the Early Stages ofPlasmodiumLiver Infection

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples

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