Transcriptome-wide dynamics of extensive m6A mRNA methylation duringPlasmodium falciparumblood-stage development

Abstract

39 40 Malaria pathogenesis results from the asexual replication of Plasmodium falciparum 41 within human red blood cells, which relies on a precisely timed cascade of gene 42 expression over a 48-hour life cycle. Although substantial post-transcriptional 43 regulation of this hardwired program has been observed, it remains unclear how 44 these processes are mediated on a transcriptome-wide level. To this end, we 45 identified mRNA modifications in the P. falciparum transcriptome and performed a 46 comprehensive characterization of N 6 -methyladenosine (m 6 A) over the course of 47 blood stage development. Using mass spectrometry and m 6 A RNA sequencing, we 48 demonstrate that m 6 A is highly developmentally regulated, exceeding m 6 A levels 49 known in any other eukaryote. We identify an evolutionarily conserved m 6 A writer 50 complex and show that knockdown of the putative m 6 A methyltransferase by 51 CRISPR interference leads to increased levels of transcripts that normally contain 52 m 6 A. In accordance, we find an inverse correlation between m 6 A status and mRNA 53 stability or translational efficiency. Our data reveal the crucial role of extensive m 6 A 54 mRNA methylation in dynamically fine-tuning the transcriptional program of a 55 unicellular eukaryote as well as a new 'epitranscriptomic' layer of gene regulation in 56 malaria parasites. 57 58 59 Keywords 60 61 Plasmodium falciparum development; mRNA methylation; N 6 -methyladenosine; 62 mRNA degradation; translational efficiency; CRISPR interference 63 64 65 66 Malaria, a mosquito-borne human disease caused by the unicellular apicomplexan 67 parasite Plasmodium falciparum, remains a major global health threat (World Health 68 Organization, 2018). Human pathogenesis results from the 48-hour intra-erythrocytic 69 developmental cycle (IDC), during which each parasite undergoes schizogony within 70 red blood cells (RBC) to create up to 32 new daughter cells (Fig. 1a). Each step of 71 the IDC -RBC invasion (0-hour post infection [hpi]), host cell remodeling, genome 72replication, and eventually RBC egress -is effected by a precisely timed cascade of 73 gene expression. Throughout this process, mRNAs from the majority of genes 74 expressed reach peak abundance only once, which is thought to correspond to the 75 time point when its encoded product is most required 1 . 76At the transcriptional level of gene regulation, silencing is achieved via 77 heterochromatinization of genes encoding variant surface antigens 2 and the inducer 78 of sexual stage development 3 . Heterochromatin, however, is restricted to 79 subtelomeric and several central chromosomal regions 4 , and the large majority of 80 the P. falciparum genome remains in a euchromatic and transcriptionally permissive 81 state throughout the IDC 5 . Recent studies of nucleosome occupancy have shown 82 that dynamic chromatin accessibility in promoter regions strongly correlates with 83 mRNA abundance, and that these sequences can be recognized by transcription 84 factors in vitro 6-8 . Intriguingly, ...

Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets

“…Numerous studies have shown that Plasmodium spp. use various post-transcriptional regulatory mechanisms to coordinate stage transitions and development (Baumgarten et al, 2019;Bunnik et al, 2016;Foth et al, 2011;le Roch et al, 2004;Tarun et al, 2008;Vembar et al, 2016). Our data corroborate these findings and suggest that similar strategies may be used in P. vivax to regulate liver stage development.…”

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

“…The Toxoplasma m6A writer complex possesses distinct features In this study, we demonstrated that the Toxoplasma m6A writer complex consists of METTL3, METTL14, WTAP, a divergent VIRMA, and a writer-associated protein (WAP1) that remains to be characterized. Recently, affinity purification of the P. falciparum METTL3 homologue revealed that the writer complexes share a similar composition in both organisms, since METTL3, METTL14, WTAP, and the unannotated VIRMA (PF3D7_1366300) were co-purified (28). The Toxoplasma m6A writer complex also shares some features of that in higher eukaryotes, such as a labile interaction between the METTL3-METTL14 and the WTAP portions of the complex.…”

“…In other systems, this occurs on specific consensus sequences, supposedly due to recognition of these motifs by components of the writer complex. While animals, plants, and brewer's yeast share similar motifs of DRACH, RRACH, and RGAC, respectively (8,41,42), these motifs are varied in parasites such as P. falciparum (GGACA) (28) and Trypanosoma brucei (CAU) (29). Using an m6A-enrichment strategy paired with high-throughput RNAseq, we identified a strongly enriched YGCAUGCR motif with a centrally-located adenosine that represents the methylated residue (Fig.…”

“…In other organisms, the distribution of m6A marks within the transcriptome are known to be enriched in specific transcript regions. For example, m6A marks tend to localize near the stop codon in mammals ( 8), within the CDS in Plasmodium falciparum (28), and within the polyA tail in Trypanosoma brucei (29). A metagene plot of m6A peaks demonstrates that they are highly enriched in Toxoplasma 3'-UTRs (Fig.…”

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

“…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.…”