Genome-wide detection of conserved stage-specific RNA editing events for nuclear genes in the Plasmodium falciparum 3D7 malaria parasite

Genome-wide detection of conserved stage-specific RNA editing events for nuclear genes in the Plasmodium falciparum 3D7 malaria parasite | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide detection of conserved stage-specific RNA editing events for nuclear genes in the Plasmodium falciparum 3D7 malaria parasite Md Thoufic Anam Azad, Tatsuki Sugi, Umme Qulsum, Kentaro Kato This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7233611/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background RNA editing is an important post-transcriptional modification of RNA. While transcriptional variation is a well-studied phenomenon in Plasmodium , post-transcriptional modifications, such as RNA editing, have not received as much scrutiny. Methods We detected genome-wide RNA editing in a developmental stage-specific manner at 16 h, 24 h, 32 h, and 40 h of tightly synchronized P. falciparum 3D7 by using the RNA editing computational approaches REDItools1-Denovo and REDItools2. Results REDItools1, the Denovo approach revealed extensive A-to-G, G-to-A, and T-to-C type variations in almost all stages. With the REDItools2 approach and screening, almost all editing events were observed in time-specific parasitic stages. G-to-A and C-to-T events were found at much higher levels. We observed significant differences in stage-specific RNA editing at 8-h intervals. RNA editing was observed at the early ring stages of the parasites, gradually increasing and then decreasing leading up to the 40-hour mature stage. Adenosine deaminase expression did not correlate with the editing level in a time-dependent manner. However, the expression of cytidine deaminase was found to be higher in the ring stages of the parasites, gradually decreasing in the later stages. The expression of other RNA editing-related factors was similar in all developmental stages. Pathways associated with RNA editing were found to be downregulated at the 40 h stage, including RNA binding, nucleic acid binding, and catalytic activity acting on RNA pathways. These findings suggest the presence of robust RNA editing machinery in Plasmodium , facilitating rapid base conversions within a short timeframe. Conclusion Our findings will be helpful in identifying the RNA editing machinery, which could serve as a potential tool for antimalarial drug discovery and malaria control in the future. DNA Gene modification mutation Time-course Variation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Malaria remains a pervasive global health issue, posing major challenges to control efforts due to its adaptability [ 1 ] (World Health Organization (WHO), 2023). Among the various Plasmodium species, Plasmodium falciparum stands out as the deadliest pathogen, surpassing the threat posed by Plasmodium vivax [ 2 ] (World Health Organization (WHO), 2022). Genetic variation is a common phenomenon observed in malaria parasites, which they employ to adapt to their hosts and vectors [ 3 ]. A Single Nucleotide Polymorphism (SNP) is a type of genetic variation that commonly occurs among individual organisms [ 4 – 6 ]; however, adopting the benefits of SNPs in the genome of an organism can be a challenging process. In contrast, transcriptional variation represents a comparatively easier genetic change and adaptive mechanism that is observed in Plasmodium and other organisms [ 7 , 8 ]. In particular, single-nucleotide variations (SNVs) occur at a much higher frequency in Plasmodium than in bacteria [ 3 , 9 ]. These SNVs in Plasmodium may arise from transcriptional errors and RNA editing processes [ 9 ]. RNA editing is a post-transcriptional mechanism that can alter nucleotides, thereby recoding the transcriptome [ 10 – 12 ]. This process can involve the deamination, amination, addition, or deletion of nucleotides by specific enzymes or enzyme systems. Among these, the most common deaminase enzymes are responsible for A-to-I RNA editing, largely mediated by the Adenosine Deaminase Acting on RNA (ADAR), and C-to-U RNA editing, mediated by the APOBEC deaminase family, in humans [ 13 , 14 ]. RNA editing has been extensively studied in animals [ 10 – 13 ]. In polar octopus, RNA editing of potassium (K+) channels is related to acclimatization to external environmental pressure [ 7 ]. In cephalopods, extensive RNA editing occurs in response to environmental pressure [ 15 ]. In the last few years, research has focused on investigating RNA editing in the nuclear genes of plants [ 16 , 17 ]. Previous studies found that the genes responsible for RNA editing in Arabidopsis thaliana undergo RNA editing themselves [ 16 ] in the seedling stages of developmental. It has been reported that RNA editing occurs in Arabidopsis thaliana at 12-day intervals, reaching up to 20 percent [ 16 – 18 ]. The phenomenon of RNA editing was first observed in trypanosome mitochondria, and its complex mechanism has recently been revealed [ 19 ]. RNA editing in the apicoplast of apicomplexan parasites has been investigated to some extent [ 20 ]. We reported on RNA editing in Plasmodium nuclear transcripts, particularly at 28S rRNA [ 21 ]. It has been reported that certain single nucleotide changes greatly affect the sickle haemoglobin dependence of the malaria parasites [ 22 ]. Plasmodium spp have Adenosine deaminase 2 (ADA2)(Fan et al., 2004) and Adenosine deaminase (ADA), which convert adenosine into inosine and the inosine functions as guanosine during translation. ADAs have different ligand binding affinities [ 23 ]. The Pf ADA-Asp176 residue is essential for conversion of adenosine and especially for 5′-methylthioadenosine (MTA) [ 24 ]. ADAs are essential enzymes for the Plasmodium survival [ 24 ]. Therefore, it is reasonable to predict that ADAs, along with other deaminases such as cytidine deaminase, may be involved in RNA editing. The multidrug resistance capacity and evolutionary mechanisms of an organism are largely influenced SNV-like genome plasticity [ 7 , 25 ]. Post-transcriptional variation is highly prevalent phenomenon in Plasmodium [ 9 ]. The effect of RNA editing on the transcriptome and proteome, and its functional implications, have been extensively studied in animals [ 7 , 26 ]. The landscape of RNA editing has been studied in other organisms on an even larger scale [ 7 , 11 , 17 , 18 , 27 – 31 ] and detailed studies in Plasmodium are lacking. Transcriptional variation has been studied to a limited extent in Plasmodium in recent years, facilitated by advancements in next-generation sequencing and large-scale data analysis, which have led to a better understanding of its adaptivity and clonal variation [ 9 ]. Additionally, SNV in Plasmodium has been observed at higher levels within its 48-hour life cycle compared to bacteria [ 9 ]. But there is still much to learn about the extent and causal factors of post-transcriptional variation, such as RNA editing in Plasmodium . RNA editing may alter the surface ligands of the parasites by altering single nucleotides. Such alterations may affect the virulence capacity of the parasites. As Plasmodium acclimatizes to the host and vector with fluctuating temperatures, we hypothesize that single nucleotide alterations by RNA editing may serve as an adaptive switch for Plasmodium spp. Here, we examined genome-wide RNA editing at 8-h intervals across three developmental stages: ring, trophozoites, and schizont of malaria parasites. We applied detailed approaches to detect developmental stage-specific RNA editing events in Plasmodium . The REDItools2 approach mainly involves comparing mRNA sequencing data with the reference genome sequence database. Grep commands were used to identify conserved editing sites and their locations with previously reported mRNA sequence variation data. We found that Plasmodium nuclear transcripts are more prone to RNA editing, and significant differences occur in a stage-specific manner. RNA-editing factors were found to positively correlate with the occurrence of RNA editing, although some factors were found to be negatively correlated. We also observed that the RNA-editing pathways positively correlated with the extent of RNA editing across different developmental stages. Materials and methods Parasite culture conditions and maintenance Plasmodium falciparum 3D7 parasites were cultured for 15 days as described previously [ 32 ]. Briefly, parasites were cultured in RPMI 1640 medium (Sigma-Aldrich) in 10-ml flasks supplemented with 25 mM HEPES, 100 µM hypoxanthine (Wako), 12.5 µg/ml gentamicin (Sigma-Aldrich), and 0.5% (w/v) Albumax-I (Invitrogen) in 5% CO 2 and N 2 at 37°C. Cell cycles were observed for one week, and early ring-stage parasites were treated with filter-sterilized (Millex GV 0.22 µm) 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.) for initial synchronization. Sorbitol-treated parasites were cultured for another week to increase their numbers with synchronized cell cycles, and the media were changed every 24 h. The culture was then diluted into another 10-ml flask to maintain approximately 5% parasitemia at 3% hematocrit. When the parasite concentration reached the desired level and the late-stage schizonts were ready to release merozoites, mature schizonts were collected using Percoll (GE Healthcare) gradient synchronization (40% and 70%). Tight synchronization of Plasmodium falciparum Early ring-stage parasites were synchronized by using 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.), whereas mature-stage parasites with higher parasitemia (15%) were synchronized by using Percoll (GE Healthcare) gradient synchronization techniques at 40% and 70% concentrations. Additionally, MT Biotech MACS MS column purification was employed to obtain highly synchronized parasites. Highly synchronized schizont-stage parasites were cultured with fresh (Read Blood Cells) RBCs for 5 h, allowing them to invade the RBCs and produce fresh ring-form parasites. After 5 h of incubation, the parasites were again synchronized by using 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.) to eliminate any remaining mature-stage parasites. The schizonts were then allowed to invade RBCs for another 5 h before being treated once more with 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.) to destroy any remaining late-stage parasites and schizonts. RNA extraction for NGS RNA was extracted from different stage parasites cultured at 8-h intervals, namely 16, 24, 32, and 40 h, to capture RNA from the ring, trophozoites, and schizont stages. RNA extraction was performed using Trizol reagent (Invitrogen) with some modifications as described previously (Moll et al., 2013). Briefly, approximately 100 µL of packed iRBCs was washed with 10x volume of sterile ice-cold PBS. Trizol reagent (Invitrogen) was added at a 10x volume ratio, followed by the addition of 200 µl of chloroform. The mixture was hand-shaken for 15 s by inverting the tube and then incubated for 2–3 minutes at room temperature. After centrifugation at 12,000 × g for 15 minutes at 4°C, the supernatant containing RNA was transferred to another tube. About 0.5 mL of ice-cold 2-propanol or 100% isopropanol were added to the aqueous phase containing RNA, followed by gentle mixing by inversion. The sample was then incubated at room temperature for 10 minutes, and then centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was discarded, and the RNA pellet was washed with 0.5 mL of ice-cold 75% ethanol. After centrifugation, the wash was discarded, and the RNA pellet was air-dried for 5–10 minutes before being resuspended in RNase-free water. The RNA was then treated with DNAase according to the manufacturer's instructions (Fast Gene RNA premium kit, Nippon Genetics Co. Ltd.). RNA quality was assessed by using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and samples were stored at -80°C until further use. Library preparation for mRNA sequencing Samples were sent to Macrogen, Japan for library preparation and total mRNA NGS. Sample initial quality control was confirmed by Macrogen and the samples proceeded to the next steps. The TruSeq stranded mRNA library preparation kit was used. Samples were sequenced in paired ends at a 101-bp read length using the NovaSeq sequencing platform. After sequencing, results were again checked and the quality measured by Macrogen, Japan. Approximately > 4 Gbp of data for each sample with a GC content > 30% and Q30 > 94% reads were obtained for each sample. MD5 values were checked for data integrity upon download. Quality control and data analyses Quality control After sequencing, initial quality control was performed by Macrogen, Japan. Quality assessment was conducted using FastQC and FastTP, considering only good quality reads for further analysis. A quality Phred score cutoff of 25 was applied for subsequent analysis steps. Trim Galore was utilized for adapter trimming and removal of low-quality reads. The version used was Trim Galore 0.6.10 with Cutadapt version 4.1, running on Python 3.8.5. The adapter sequence 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR) was removed. Paired-end trimming mode was employed, resulting in approximately 33% of reads containing adapters, with all reads passing filters. Data analyses STAR alignments were performed to align the paired-end clean reads with the reference genome of Plasmodium falciparum 3D7. We utilized STAR version 2.7.10b, for this purpose. BAM files were generated, and Samtools was employed to create a BAM index file for subsequent analysis. Command-lines are available in the supplementary data (Supplementary data 1). Expression of RNA editing-related genes Raw reads were further analyzed by using iDEP [ 33 ]. Raw read counts were uploaded to the iDEP interface, and the reference genome of Plasmodium falciparum was selected or automatically matched to the best matching Plasmodium falciparum genome. Dseq1 and Dseq2 analyses were performed via the iDEP interface. The raw read data were also analyzed using iDEP v1.0 [ 33 ]. The data were initially filtered to remove reads below 0.5 CPM in at least one sample. Then, the data were transformed using EdgeR with a pseudocount of 4, and missing values were imputed using gene median. Of the 5767 genes in 12 samples (three replicates for each), 5397 passed the filter. These 5397 genes were converted to Ensembl gene IDs. Transformed group expressions were further analyzed by using iDEP1.1 [ 33 ]. Three biological replicates for each condition were included in the analyses. RNA variation detection by REDItools1, Denovo approach REDItools1, using a denovo approach, primarily compares RNA sequencing data from different experimental conditions. Reference genomic and mRNA sequences are not essential for this analysis. However, we utilized the reference genomic sequence of Plasmodium falciparum 3D7 for the better analysis. The parameters used include a minimum per base coverage of 10, a minimum per base quality of 30 (specified by the -q option), a significance value of 0.05, and a minimum variation frequency of 0.10. RNA editing detection by REDItools2 The REDItools2 approach involves comparing mRNA sequencing data with either the reference genome or a genomic sequence database. In this study, we utilized the reference genome of Plasmodium falciparum 3D7. Different RNA editing events were counted in a stage-specific manner using the REDItools output tables, with three biological replicates analyzed. Grep commands were employed to identify editing locations with reference nucleotide variation data, excluding SNPs. Exploring pathways associated with RNA editing Gene Ontology (GO) pathways were analyzed by using iDEP resources [ 33 ]. Molecular pathways were examined among the three pathways assessed. We observed that RNA editing-related molecular pathways were predominantly downregulated at the 40-h stage compared to the other parasitic stages. The interrelated pathway was also analyzed by using iDEP resources [ 33 ]. The degree of relationship between different RNA modification pathways and their expression was assessed. Results Flow chart of the study A visual representation of the processes and workflow involved in this study are presented in the flow chart (Flow chart 1). Sample preparation and data analysis PCA (principal component analysis) indicated that samples from the same time course clustered together (Additional file 1 Fig. 1 A). Clustering of the heat map of 2100 genes also indicated that the sample preparation was suitable for further analysis of the RNA sequence data (Additional file 1 Fig. 1 B). RNA variation detection by REDItools1, Denovo approach REDItools1, Denovo approach provides the variation among RNA sequence data with or without comparing to a reference sequence. Here, we compared three replicates of RNA sequencing data with the reference Plasmodium genome. A-to-G, G-to-A, T-to-A, T-to-C, and C-to-T variation was detected at a much higher level than the other variations by using REDItools1; denovo approach (Fig. 1 ). By using the REDItools 1 denovo approach, we found that the A-to-G and G-to-A conversions were almost similar. A-to-G events at 16 h, 24 h, 32 h, and 40 h were found at 84497, 85847, 87897, and 108198 sites, respectively (Fig. 1 A). G-to-A events at 16 h, 24 h, 32 h, and 40 h were found at 74988, 76731, 81508, and 88817 sites, respectively. C-to-T events at 16 h, 24 h, 32 h, and 40 h were found at 44952, 41313, 40223, and 47903 sites, respectively. T-to-C variations at 16 h, 24 h, 32 h, and 40 h were found at 67737, 63551, 62792, and 71170 sites, respectively. The proportions of A-to-G, G-to-A, C-to-T, and T-to-A RNA variations were 18%, 16%, 8%, and 11%, respectively (Fig. 1 B). In this approach, the pattern of modification was almost the same except for the number of base modifications or variations (Additional file 1 Fig. 2 ). RNA editing detection by REDItools2 We identified the conserved RNA editing events in Plasmodium and detected how the process continues during the Plasmodium life cycle. The reference data of RNA variation were used in grep command with our data tables to identify the conserved RNA-editing types, locations, and numbers. In analyses using REDItools 2, the frequency of G-to-A editing was found to be higher than that of A-to-G editing. This finding indicates that the amination reaction is more frequent than the deamination-type RNA editing. C-to-T was higher than T-to-C. In this case, deamination-type RNA editing was more frequent than the amination-type RNA editing. A-to-G editing events at 16 h, 24 h, 32 h, and 40 h were found 60, 66, 71, and 58 sites, respectively. G-to-A editing events at 16 h, 24 h, 32 h, and 40 h were found at 217, 236, 259, and 213 sites, respectively. C-to-T editing events at 16 h, 24 h, 32 h, and 40 h were found at 194, 220, 246, and 200 sites, respectively (Fig. 2 ). T-to-C editing events at 16 h, 24 h, 32 h, and 40 h were found at 55, 64, 66, and 65 sites, respectively. In our study using REDItools2 and strict screening, we detected various types of editing, including C-to-G, T-to-G, A-to-T, A-to-G, G-to-A, T-to-C, and A-to-C transitions. Among these, deamination-type RNA editing, A-to-G and C-to-T, was found at frequencies of 5% and 17%, respectively. G-to-A and T-to-C occurred at frequencies of 18% and 5%, respectively (Fig. 2 ), and represent rare events of amination-type RNA editing in animals and plants. Almost all types of RNA editing started in early developmental, that is, with ring-stage parasites, and gradually increased with for trophozoites and then declined significantly for schizont-stage parasites (Fig. 3 ). From 16 h to 32 h, there was a significant increase in RNA editing events (Fig. 3 ). Conversely, from 32 h to 40 h, there was a significant decrease in RNA editing events. These findings indicate that RNA editing starts at the early ring stages of the parasite and gradually decreases as the parasite reaches maturity. In the REDItools2 analyses, samples from the batch 1, batch 2, and batch 3 replicates exhibited the same pattern and extent of RNA editing (Additional file 1 Fig. 3 ). The genome-wide editing event patterns were almost identical across all parasitic stages in the three biological replicates (Additional file 1 Fig. 4 ). Expression of RNA editing-related genes ADA2, ADA, Cytidin Deaminase, PPR, cytidin and deoxycytidylate deaminase, and PPR1 have previously been implicated in RNA editing [ 16 , 23 , 34 – 36 ]. These genes are homologues RNA-editing genes in other species including higher animals and plants. The expression patterns of these genes are presented in Fig. 4 A. ADA2 and ADA were found in almost all parasitic stages but their expression did not correlate with the editing pattern, which was higher in the mature parasitic stages. Cytidine deaminase expression was found to be higher in the ring parasitic stages but gradually decreased in the late stages of the parasite. The expression of cytidine deaminase was negatively correlated with the editing pattern in Plasmodium . Cytidine and deoxycytidylate deaminase also positively correlated, to some extent, with RNA editing, but to a lesser extent than cytidine deaminase. The expression of cytidine and deoxycytidylate deaminase slightly increased in the ring stages and decreased in the late parasitic stages. In addition, the expression of homologues of PPR and PPR1, important enzyme families in plants, were negatively correlated with the editing pattern (Fig. 4 A). In contrast, the expression of PPR and PPR1, proteins responsible for RNA binding and modification, decreased in the ring stage and increased in the later parasitic stages. Expression of Zinc fingure genes Some Zinc fingure genes have been reported to be involved in RNA editing [ 34 , 37 – 39 ]. We studied the genes PF3D7_1209300 and PF3D7_1009400, which belong to the C2H2 subfamily; PF3D7_1419900, which belongs to the CCCH subfamily; PF3D7_0627300, PF3D7_1422500, PF3D7_1235300, PF3D7_0512300, and PF3D7_1014600, which belong to an RFP subfamily; PF3D7_1008100, PF3D7_1433400, PF3D7_0629700, and PF3D7_1322100, which belongs to the PHD subfamily; and PF3D7_0314700 and PF3D7_1221000, which belongs to another RFP subfamily. High expression of PF3D7_1009400, PF3D7_0627300, PF3D7_1433400; PF3D7_0629700, and PF3D7_1235300 was found in almost all stages. The expression of PF3D7_1209300, PF3D7_1422500, and PF3D7_1221000 started in the ring stage and gradually increased in the later parasitic stages (Fig. 4 B). In contrast, PF3D7_1419900, PF3D7_1235300, PF3D7_0512300, and PF3D7_1322100 expression was high at the ring stage and decreased gradually during the later stages of parasitic development (Fig. 4 B). Expression of the other genes was almost similar in the ring, trophozoites, and schizont stages of the parasites. Pathways related to RNA editing In the comparisons between 40 h-16 h, 32 h-16 h, and 24 h-16 h, 1930, 1655, and 807 genes were downregulated, respectively. The downregulated pathways include RNA binding, nucleic acid binding, and catalytic activity acting on RNA (Fig. 5 , Additional file 1 Fig. 6 A). The RNA binding pathway is particularly important for RNA editing. The 50 most upregulated gene numbers were increased at the 40-h parasite stage. However, we observed a gradual downregulation of the RNA binding pathway up to the 40-h stage of the parasite (Supplemen Additional file 1 tary Fig. 6 B). In the comparison between the 24 h and 16 h stages, pathways probably related to RNA editing were not found to be downregulated in the 24 h stage parasites (Fig. 5 ). Analyses of the relationships among the enriched Gene Ontology (GO) molecular function terms, including RNA binding, nucleic acid binding, and catalytic activity acting on RNA pathways, indicated that these downregulated pathways are related to RNA-editing factors. In the analyses of the network of interrelated pathways, we observed that in the 40 h-32 h comparison, the most downregulated pathways were nucleic acid binding and catalytic activity acting on RNA pathways. Moreover, the catalytic activity acting on RNA pathway shares genes with the RNA methyl transferase activity pathway (Fig. 5 ). In the 40 h-16 h comparison, the highly downregulated pathways included RNA binding pathways containing 292 genes, nucleic acid binding pathways containing 383 genes, and catalytic activity acting on RNA pathways containing 147 genes. In the 32 h-16 h comparison, the moderately downregulated pathways consisted of RNA binding pathways containing 195 genes, nucleic acid binding pathways containing 269 genes, and SnoRNA binding pathways containing only 13 genes. For the 24 h-16 h parasite stage comparison, the highly downregulated pathways were host cell surface binding and cell adhesion molecule binding pathways. Interestingly, pathways related to RNA editing showed no downregulation in the 24 h ring-stage parasites when compared to the 16 h stage (Fig. 5 ). In contrast, in the 40 h-16 h comparison, the most downregulated pathway at 40 h was the RNA binding pathway. In the 40 h-16 h, 40 h-24 h, and 40 h-32 h comparisons, 821 genes were commonly downregulated in all groups and downregulated in the 40-h samples. However, in the 24 h-16 h, 32 h-24 h, and 40 h-32 h stage comparisons, only 61 genes were commonly downregulated at the 24 h, 32 h, and 40 h stages (Fig. 6 B). Of 20 genes tested, those most significantly affected were found to be downregulated at the 40-h stage (Additional file 1 Fig. 5 ). Furthermore, the RNA binding pathway shared common genes with the heterocyclic compound binding, nucleic acid binding, and organic cyclic compound binding pathways (Fig. 6 C and D). Discussion RNA editing is an important post-transcriptional phenomenon in animal, plants, and microorganisms that increase proteome and transcript diversity. Here, we investigated genome-wide RNA editing that contributed to transcriptional variation. When we utilized the REDItools Denovo approach, we found a high number of SNVs. Previous reports have indicated that the highest numbers of variations in Plasmodium involve A-to-G, T-to-C, and A-to-T substitutions. Among these, A-to-G and C-to-T SNVs comprise approximately 28% and 4%, respectively [ 9 ]. In the Denovo approach, we found that A-to-G and G-to-A conversions occurred in almost similar proportions. This observation suggests that both deamination and amination types of conversions occur in Plasmodium at similar rates. We then employed the REDItool2 approach and screened out the SNPs and selected the conserved RNA editing sites by comparing them with previously identified nucleotide variation sites genome-wide [ 9 ]. Next, we screened out the SNPs and selected only the conserved RNA editing sites between two laboratory-adapted Plasmodium species. We used the grep command and observed a significant reduction in the number of RNA editing events, decreasing from thousands to hundreds. The most common type of RNA editing events were A-to-T, G-to-A, T-to-A, and C-to-T. We found a higher level of C-to-T type editing, and the enzyme responsible for this conversion is cytidine deaminase. Of note, we used highly synchronized Plasmodium so we could observe stage-specific events. A-to-G and C-to-T RNA editing usually happen in humans and animals [ 30 , 40 – 42 ]. In plants, T-to-C type editing occurs [ 17 , 18 ]. In our study, the editing types in Plasmodium are more diversified than those reported for other species. We observed a significant difference in the total number of RNA editing events across the developmental stages of Plasmodium falciparum 3D7 parasites. Such variation may help parasites acclimate to the temperature fluctuations in the host. These types of significant changes were observed for only 8-h time intervals, indicating that the RNA editing starts at the early ring stage of the parasites, increases during the trophozoite stage, and gradually decreases when the parasites reach the schizont stage. Genome-wide editing events were almost identical in all parasitic stages across the three biological replicates. Similar developmental stage-specific phenomena have also been observed in Xenopus [ 43 ]. In our study, we detected a significant level of RNA editing occurring within a short 8-h period. Editing begins during the early ring parasitic stages, increases up to the 32 h stage, and then decreases significantly. These findings suggest that stage-specific RNA modification is an important phenomenon in this parasite. In our previous study in HEK-293 cells, we observed a considerable level of RNA editing occurring with engineered human deaminase enzymes within a short period [ 21 , 44 , 45 ]. It has recently been reported that Adenosine Deaminase Acting on RNA (ADARs) have evolved in birds to function optimally at 40°C, targeting different groups of temperature-sensitive RNAs [ 46 ]. During the early ring stages and the late schizonts stage, prior to the organism encountering the higher core body temperature of its host, the recognition of Plasmodium RNA by ADARs complementary factors may be altered. Temperature may affect the expression of the RNA-editing factors and the structure of the RNA [ 26 ]. Hence, RNA editing may play a vital role in the rapid and intermittent adaptation of malaria organisms to higher host core body temperatures. In most previous reports, RNA editing has been found to be linked to the expression of RNA-editing enzymes and tissue specificity [ 47 , 48 ]. The editing enzymes are mostly deaminases. In plants, PPR [ 35 ] and zinc finger proteins are also involved in RNA editing [ 34 ]. The ADA gene is essential for malaria parasite survival and adenosine modification in Plasmodium [ 23 , 24 ]. Adenosine conversion by ADA has been reported in vitro [ 24 ]. High levels of cytidine deamination are observed with higher expression of cytidine deaminase in the ring stage of parasites, and animal and plant deaminase gene expression is usually proportional to RNA editing [ 29 ]. Hence, in malaria parasites, ADA and other groups of genes may be involved in RNA modification, RNA editing, and parasite adaptation. Of note, the substrate recognition of ADA in Plasmodium falciparum and Plasmodium vivax differs significantly [ 24 ] . More than 30 Zinc-finger family protein are present in Plasmodium . These genes are responsible for nucleic acid binding and RNA modification [ 37 ]. In plants, representatives of the zinc-finger family protein are involved in RNA binding, RNA modification, and RNA editing processes [ 38 , 39 , 49 ]. In Plasmodium , the presence of ADA deaminase enzymes, PPR proteins, and cytidine deaminase has been reported [ 23 , 24 , 37 , 50 ]. Other genes responsible for RNA modification in Plasmodium need to be identified, as genome-wide RNA editing has been observed throughout the parasitic developmental stages. Here, we found that the expression of ADA is consistent throughout the parasitic developmental stages. Another possibility is that there are complementary RNA editing factors related to Plasmodium RNA editing that have not yet been reported. The RNA editing system of Plasmodium might resemble the plant RNA editing system, which comprises a combination of different editosome complexes [ 49 ]. It was previously assumed that the malaria organism undergoes numerous transcriptional errors during its multiplication and adaptation to various environments. However, the organism is well-equipped to maintain its high AT-rich genome, comprising approximately 70%, with its transcriptional machinery [ 51 ]. It can tightly control every step of gene regulation, chromatin structure [ 52 , 53 ] transcription [ 54 , 55 ] and translation, [ 56 , 57 ] all of which are essential for its adaptation and survival in diverse host environments. In this study, we identified a correlation between the number of RNA editing events and the pathways related to RNA editing. In the 40-h samples, most of the pathways related to RNA editing were highly downregulated and the number of RNA editing events decreased significantly. In the 32-h stage parasites, the major pathway related to RNA editing and the catalytic activity acting on RNA were not downregulated. Furthermore, other pathways related to RNA editing were less downregulated and contained fewer genes compared to the 40-h trophozoite-stage parasites. Based on these findings, we conclude that these pathways and genes are likely to be related to RNA editing as a developmental phenomenon in Plasmodium parasites. In the 40 h to 16 h stages, the number of RNA editing events was lower than in the other two stages, suggesting that the pathway involving catalytic activity acting on RNA may contain genes responsible for RNA editing. RNA-editing factors bind in the editing sites of the RNA. These editing sites are mainly defined based on the binding activity of the enzyme or enzyme complex responsible for the RNA editing [ 29 ]. The substrate recognition groove of the ADA enzyme in Plasmodium is diversified [ 24 ]. Plasmodium ADA not only binds to adenosine (A) but also has a binding pocket for inosine (I), which acts as a homologue of guanosine (G) [ 24 ]. This indicates that ADA may be responsible for both deamination and amination. Therefore, it is possible that both deaminase and aminase types of enzyme systems, along with other editosome complexes, may be present in Plasmodium . It has been reported that RNA editing is not conserved even at the species level [ 43 ]. This suggest that it may be an adaptive mechanism of the organism to its environment [ 26 , 58 ]. Our study revealed that genome-wide RNA editing contributes to transcript diversity in different developmental stages. The life cycle of Plasmodium falciparum is 48 h, during which time the enzymatic activity of the various components of the RNA modification machinery encoded in the parasite genome may be critical. Recently, it has been reported that extensive temperature-responsive reversible RNA editing phenomena occur within hours in octopus neural cells, leading to functional diversity in motor proteins such as kinesin and synaptotagmin [ 26 ]. Additionally, a stage-specific environmental stress-responsive RNA editing strategy has been identified in fungi [ 59 ]. In ducks and hummingbirds, elevated core body temperature shapes their deaminase system to recognize temperature-sensitive RNA, thus enabling them to adapt to their environment [ 46 ]. Tissue-specific temperature-dependent RNA editing has also been reported in zebrafish [ 48 ]. RNA editing might, therefore, be an important mechanism of acclimatization in the malarial pathogen, similar to these other organisms [ 7 , 8 , 26 , 48 , 60 , 61 ]. Accordingly, we believe that RNA editing plays a vital role in the biology and adaptation of the malaria parasite. It has been reported that single nucleotide alterations in Plasmodium spp. can lead to antimalarial drug resistance [ 62 ]. Temperature plays a critical role in shaping RNA and RNA editing [ 26 , 46 , 48 , 60 , 63 ]. Malaria parasites are exposed to different temperature within the host and various systems. By utilizing the RNA editing machinery, the organism may shape it transcriptomes. Malaria organisms have adapted to a wide range of hosts and to different environmental pressures. RNA editing may play a vital role in adaptation, especially to the transient and intermittent febrile temperatures of the host. Conclusions This study represents the first comprehensive investigation of genome-wide, stage-specific RNA editing in Plasmodium nuclear transcripts. We identified conserved RNA editing sites in Plasmodium falciparum 3D7, shedding light on the presence of RNA editing events throughout the Plasmodium developmental cycle. The factors involved and the targeted RNA require further investigation across Plasmodium species. Understanding heat stress adaptation, multiplication, and the factors related to RNA editing will be crucial for controlling this dangerous, drug-resistant pathogen. Our findings reveal RNA editing as a developmental phenomenon, with genome-wide conserved events detected at 8-h intervals. We observed a higher number of editing events during the ring to trophozoite developmental stages within human red blood cells, despite the short time intervals between the different developmental stages in malaria parasites. It is challenging to differentiate RNA editing differences due to these short intervals. However, it is important to note that the pipeline used in this study may have excluded certain RNA editing sites from the representative results due to limitations of the grep command. Further studies employing single-cell genome-wide transcriptomic sequencing and single-cell long-read transcriptomic sequencing will provide more detailed and conclusive insights into RNA editing in Plasmodium . More invasive and focused approaches, such as gene-specific and long-read sequencing, are needed to investigate RNA editing and its consequences in malaria parasites. A detailed understanding of such adaptive strategies may provide fundamental insights for drug and vaccine development targeting this deadly pathogen. Declarations Ethical approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Data available on request. Competing Interest The authors declare no conflicts of interest Funding This work was supported by JSPS Fellows (22F21389), and Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan, and by a Livestock Promotional Subsidy from the Japan Racing Association. Author Contributions Conceptualization, M.T.A.A. and U.Q.; methodology, M.T.A.A.; software, M.T.A.A. and T.S.; validation, M.T.A.A.; formal analysis, M.T.A.A.; investigation, M.T.A.A.; resources, K.K.; data curation, M.T.A.A.; writing—original draft preparation, M.T.A.A. and U.Q.; writing—review and editing, M.T.A.A., K.K. and U.Q.; supervision, K.K.; project administration, K.K.; and funding acquisition, K.K. and M.T.A.A. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable References World Health Organization (WHO). World malaria report 2023. Geneva: World Health Organization; 2023. World Health Organization (WHO). 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Dynamic response of RNA editing to temperature in drosophila. BMC Biol. 2015;13. Additional Declarations No competing interests reported. Supplementary Files Flowchart1.jpeg Flow chart 1. Outline of the steps involved in this study. Supplementarydata1.docx Additionalfile1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7233611","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492845207,"identity":"3852c529-3c87-4500-9c4a-38aff2a2fb6b","order_by":0,"name":"Md Thoufic Anam Azad","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"Thoufic Anam","lastName":"Azad","suffix":""},{"id":492845210,"identity":"32219a9d-3844-4269-a184-a644bd5e2962","order_by":1,"name":"Tatsuki Sugi","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Tatsuki","middleName":"","lastName":"Sugi","suffix":""},{"id":492845213,"identity":"88e7aec8-9843-4061-b811-81e71c2c0cf0","order_by":2,"name":"Umme Qulsum","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Umme","middleName":"","lastName":"Qulsum","suffix":""},{"id":492845216,"identity":"c96e6ca1-8713-47bc-9945-74b76968f5bc","order_by":3,"name":"Kentaro Kato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHUlEQVRIie2RP2uDQBiHXxHicpD1FYt+BQ+hk6VfRRF0MWunDIaALhe6Wvol2r3QCwGziPkKSiBThnYsFFo1LYRyFrKFcM9w9zuO533vD4BEcobgn/BlAhBQksPKG1AO+whqN6kOOVnx2ZEiRM8W6/rjxW3DZlnDdBS9aotl85CCNU5gVwsUg1T+jO3CNgSqDQWZMFIF9DkFmnOIbIFiYkwTwldgQjBCJcEJw/hab1JQngBCFCnWns4+O2W87RQ7Ij/K7ZBiIKHzrouBfRfP65X2YP6QorOYPl7xkOj51rG9glNWlg7NKwzylfguuC7t9z13Tdz4Tf025ZaWMdqwO/fmPmOh6MV+If3oHdVq/4iE/xhitOJkRSKRSC6RbyLBW4soPFYeAAAAAElFTkSuQmCC","orcid":"","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Kentaro","middleName":"","lastName":"Kato","suffix":""}],"badges":[],"createdAt":"2025-07-28 11:53:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7233611/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7233611/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88021991,"identity":"817abb3e-1706-4a26-9426-344908fbabd8","added_by":"auto","created_at":"2025-07-31 14:01:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118640,"visible":true,"origin":"","legend":"\u003cp\u003eRNA variation types detected by using REDItools denovo approach. A. Numbers of variations. B. Proportions of variations.\u003c/p\u003e","description":"","filename":"Fig.1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/23a65884818e21d769269032.jpeg"},{"id":88021992,"identity":"c44000b5-501c-4e9e-abc8-9ddef07fca78","added_by":"auto","created_at":"2025-07-31 14:01:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117525,"visible":true,"origin":"","legend":"\u003cp\u003eRNA editing detection by the REDItools2 approach A. REDItools2-detected RNA editing number. B. Proportions of the different kinds of RNA editing detected by REDItools2 analysis.\u003c/p\u003e","description":"","filename":"Fig.2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/090277a922cd50a21cf2257e.jpeg"},{"id":88022894,"identity":"eab7d75b-d0f1-4695-99e9-4af91cc3c040","added_by":"auto","created_at":"2025-07-31 14:09:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34297,"visible":true,"origin":"","legend":"\u003cp\u003eStage-specific RNA editing numbers in \u003cem\u003ePlasmodium falciparum \u003c/em\u003e3D7. Number of RNA editing events significantly downregulated at 8-h intervals.\u003c/p\u003e","description":"","filename":"Fig.3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/b1b3367d09cc00e6c4df8110.jpeg"},{"id":88023477,"identity":"5f0b575f-4ff7-44ec-ab33-70a74e478738","added_by":"auto","created_at":"2025-07-31 14:17:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":293380,"visible":true,"origin":"","legend":"\u003cp\u003eStage-specific expression of RNA editing related genes A. Zinc finger family gene expression. B. ADA2, ADA, Cytidin Deaminase, PPR, cytidine, and deoxycytidylate deaminase, PPR1.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/f2500c151c71d32875e83239.jpg"},{"id":88022000,"identity":"405c5b36-2d18-45d5-b094-bba4a4775e3e","added_by":"auto","created_at":"2025-07-31 14:01:56","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":153761,"visible":true,"origin":"","legend":"\u003cp\u003eStage-specific gene expression and downregulation of molecular pathways. \u0026nbsp;40 h-16 h, 32 h-16 h, and 24 h-16 h comparison showed 1930, 1655, and 807 genes were downregulated, respectively. The downregulated pathways were RNA binding, Nucleic acid binding, and Catalytic activity acting on RNA. The RNA binding pathway was an important pathway for RNA editing. The RNA binding pathway was gradually downregulated to the 40-h stage of the parasite. The 24 h-16 h stage pathways are probably involved in RNA editing that does not involve downregulation.\u003c/p\u003e","description":"","filename":"Fig.5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/3fd58816e8d9f49c6514a7d8.jpeg"},{"id":88022897,"identity":"86ee99d7-5867-4216-bc7e-ad5e1a194940","added_by":"auto","created_at":"2025-07-31 14:09:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":123161,"visible":true,"origin":"","legend":"\u003cp\u003eStage-specific gene expression and relation of different pathways. A. About 61 genes were downregulated at the 40-, 32-, and 24-h stages. These genes might play a key role in RNA editing in \u003cem\u003ePlasmodium\u003c/em\u003eincluding in adaptation such as higher temperature adaptation. C. Network of 40 h-16 h interrelated pathways. These pathways were downregulated at the 40-h stage. Two pathways are connected if they share 30% or more genes. The color depth indicates the degree of downregulation. The depth of the connected lines corresponds to the degree of shared genes. D. Molecular Network 40 h-32 h downregulated at 40 h.\u003c/p\u003e","description":"","filename":"Fig.6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/25a4508fac1f83a73da2d821.jpeg"},{"id":90326751,"identity":"11bb6995-5560-4617-8f0d-e91061a67182","added_by":"auto","created_at":"2025-09-01 12:17:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1676014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/dffaeec4-e802-470b-b318-8abad84f504b.pdf"},{"id":88021994,"identity":"1449fb1f-5333-439f-83d8-3cea4fb87e83","added_by":"auto","created_at":"2025-07-31 14:01:56","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":168912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow chart 1.\u003c/strong\u003e Outline of the steps involved in this study.\u003c/p\u003e","description":"","filename":"Flowchart1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/b6a9783f6874dbf9e641f8bf.jpeg"},{"id":88021999,"identity":"6d8bbd39-d897-4f82-8a49-d4833b250e54","added_by":"auto","created_at":"2025-07-31 14:01:56","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18320,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/90058e02e0d2e5a5486fcab1.docx"},{"id":88021996,"identity":"3261fbc5-85f2-405c-892c-95a450a514d4","added_by":"auto","created_at":"2025-07-31 14:01:56","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2215600,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7233611/v1/0e9e9ff179ddefa1337e663e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide detection of conserved stage-specific RNA editing events for nuclear genes in the Plasmodium falciparum 3D7 malaria parasite","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria remains a pervasive global health issue, posing major challenges to control efforts due to its adaptability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] (World Health Organization (WHO), 2023). Among the various \u003cem\u003ePlasmodium\u003c/em\u003e species, \u003cem\u003ePlasmodium falciparum\u003c/em\u003e stands out as the deadliest pathogen, surpassing the threat posed by \u003cem\u003ePlasmodium vivax\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] (World Health Organization (WHO), 2022). Genetic variation is a common phenomenon observed in malaria parasites, which they employ to adapt to their hosts and vectors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A Single Nucleotide Polymorphism (SNP) is a type of genetic variation that commonly occurs among individual organisms [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]; however, adopting the benefits of SNPs in the genome of an organism can be a challenging process. In contrast, transcriptional variation represents a comparatively easier genetic change and adaptive mechanism that is observed in \u003cem\u003ePlasmodium\u003c/em\u003e and other organisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In particular, single-nucleotide variations (SNVs) occur at a much higher frequency in \u003cem\u003ePlasmodium\u003c/em\u003e than in bacteria [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These SNVs in \u003cem\u003ePlasmodium\u003c/em\u003e may arise from transcriptional errors and RNA editing processes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRNA editing is a post-transcriptional mechanism that can alter nucleotides, thereby recoding the transcriptome [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This process can involve the deamination, amination, addition, or deletion of nucleotides by specific enzymes or enzyme systems. Among these, the most common deaminase enzymes are responsible for A-to-I RNA editing, largely mediated by the Adenosine Deaminase Acting on RNA (ADAR), and C-to-U RNA editing, mediated by the APOBEC deaminase family, in humans [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRNA editing has been extensively studied in animals [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In polar octopus, RNA editing of potassium (K+) channels is related to acclimatization to external environmental pressure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In cephalopods, extensive RNA editing occurs in response to environmental pressure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the last few years, research has focused on investigating RNA editing in the nuclear genes of plants [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies found that the genes responsible for RNA editing in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e undergo RNA editing themselves [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] in the seedling stages of developmental. It has been reported that RNA editing occurs in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e at 12-day intervals, reaching up to 20 percent [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The phenomenon of RNA editing was first observed in trypanosome mitochondria, and its complex mechanism has recently been revealed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRNA editing in the apicoplast of apicomplexan parasites has been investigated to some extent [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We reported on RNA editing in \u003cem\u003ePlasmodium\u003c/em\u003e nuclear transcripts, particularly at 28S rRNA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has been reported that certain single nucleotide changes greatly affect the sickle haemoglobin dependence of the malaria parasites [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003ePlasmodium\u003c/em\u003e spp have Adenosine deaminase 2 (ADA2)(Fan et al., 2004) and Adenosine deaminase (ADA), which convert adenosine into inosine and the inosine functions as guanosine during translation. ADAs have different ligand binding affinities [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The \u003cem\u003ePf\u003c/em\u003eADA-Asp176 residue is essential for conversion of adenosine and especially for 5\u0026prime;-methylthioadenosine (MTA) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. ADAs are essential enzymes for the \u003cem\u003ePlasmodium\u003c/em\u003e survival [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, it is reasonable to predict that ADAs, along with other deaminases such as cytidine deaminase, may be involved in RNA editing.\u003c/p\u003e\u003cp\u003eThe multidrug resistance capacity and evolutionary mechanisms of an organism are largely influenced SNV-like genome plasticity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Post-transcriptional variation is highly prevalent phenomenon in \u003cem\u003ePlasmodium\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The effect of RNA editing on the transcriptome and proteome, and its functional implications, have been extensively studied in animals [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The landscape of RNA editing has been studied in other organisms on an even larger scale [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and detailed studies in \u003cem\u003ePlasmodium\u003c/em\u003e are lacking. Transcriptional variation has been studied to a limited extent in \u003cem\u003ePlasmodium\u003c/em\u003e in recent years, facilitated by advancements in next-generation sequencing and large-scale data analysis, which have led to a better understanding of its adaptivity and clonal variation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, SNV in \u003cem\u003ePlasmodium\u003c/em\u003e has been observed at higher levels within its 48-hour life cycle compared to bacteria [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBut there is still much to learn about the extent and causal factors of post-transcriptional variation, such as RNA editing in \u003cem\u003ePlasmodium\u003c/em\u003e. RNA editing may alter the surface ligands of the parasites by altering single nucleotides. Such alterations may affect the virulence capacity of the parasites. As \u003cem\u003ePlasmodium\u003c/em\u003e acclimatizes to the host and vector with fluctuating temperatures, we hypothesize that single nucleotide alterations by RNA editing may serve as an adaptive switch for \u003cem\u003ePlasmodium\u003c/em\u003e spp.\u003c/p\u003e\u003cp\u003eHere, we examined genome-wide RNA editing at 8-h intervals across three developmental stages: ring, trophozoites, and schizont of malaria parasites. We applied detailed approaches to detect developmental stage-specific RNA editing events in \u003cem\u003ePlasmodium\u003c/em\u003e. The REDItools2 approach mainly involves comparing mRNA sequencing data with the reference genome sequence database. Grep commands were used to identify conserved editing sites and their locations with previously reported mRNA sequence variation data. We found that \u003cem\u003ePlasmodium\u003c/em\u003e nuclear transcripts are more prone to RNA editing, and significant differences occur in a stage-specific manner. RNA-editing factors were found to positively correlate with the occurrence of RNA editing, although some factors were found to be negatively correlated. We also observed that the RNA-editing pathways positively correlated with the extent of RNA editing across different developmental stages.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eParasite culture conditions and maintenance\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7 parasites were cultured for 15 days as described previously [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, parasites were cultured in RPMI 1640 medium (Sigma-Aldrich) in 10-ml flasks supplemented with 25 mM HEPES, 100 \u0026micro;M hypoxanthine (Wako), 12.5 \u0026micro;g/ml gentamicin (Sigma-Aldrich), and 0.5% (w/v) Albumax-I (Invitrogen) in 5% CO\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Cell cycles were observed for one week, and early ring-stage parasites were treated with filter-sterilized (Millex GV 0.22 \u0026micro;m) 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.) for initial synchronization. Sorbitol-treated parasites were cultured for another week to increase their numbers with synchronized cell cycles, and the media were changed every 24 h. The culture was then diluted into another 10-ml flask to maintain approximately 5% parasitemia at 3% hematocrit. When the parasite concentration reached the desired level and the late-stage schizonts were ready to release merozoites, mature schizonts were collected using Percoll (GE Healthcare) gradient synchronization (40% and 70%).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTight synchronization of\u003c/b\u003e \u003cb\u003ePlasmodium falciparum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEarly ring-stage parasites were synchronized by using 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.), whereas mature-stage parasites with higher parasitemia (15%) were synchronized by using Percoll (GE Healthcare) gradient synchronization techniques at 40% and 70% concentrations. Additionally, MT Biotech MACS MS column purification was employed to obtain highly synchronized parasites. Highly synchronized schizont-stage parasites were cultured with fresh (Read Blood Cells) RBCs for 5 h, allowing them to invade the RBCs and produce fresh ring-form parasites. After 5 h of incubation, the parasites were again synchronized by using 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.) to eliminate any remaining mature-stage parasites. The schizonts were then allowed to invade RBCs for another 5 h before being treated once more with 5% D-sorbitol (Tokyo Chemical Industry Co., Ltd.) to destroy any remaining late-stage parasites and schizonts.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA extraction for NGS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA was extracted from different stage parasites cultured at 8-h intervals, namely 16, 24, 32, and 40 h, to capture RNA from the ring, trophozoites, and schizont stages. RNA extraction was performed using Trizol reagent (Invitrogen) with some modifications as described previously (Moll et al., 2013). Briefly, approximately 100 \u0026micro;L of packed iRBCs was washed with 10x volume of sterile ice-cold PBS. Trizol reagent (Invitrogen) was added at a 10x volume ratio, followed by the addition of 200 \u0026micro;l of chloroform. The mixture was hand-shaken for 15 s by inverting the tube and then incubated for 2\u0026ndash;3 minutes at room temperature. After centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 minutes at 4\u0026deg;C, the supernatant containing RNA was transferred to another tube. About 0.5 mL of ice-cold 2-propanol or 100% isopropanol were added to the aqueous phase containing RNA, followed by gentle mixing by inversion. The sample was then incubated at room temperature for 10 minutes, and then centrifuged at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C. The supernatant was discarded, and the RNA pellet was washed with 0.5 mL of ice-cold 75% ethanol. After centrifugation, the wash was discarded, and the RNA pellet was air-dried for 5\u0026ndash;10 minutes before being resuspended in RNase-free water. The RNA was then treated with DNAase according to the manufacturer's instructions (Fast Gene RNA premium kit, Nippon Genetics Co. Ltd.). RNA quality was assessed by using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and samples were stored at -80\u0026deg;C until further use.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLibrary preparation for mRNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSamples were sent to Macrogen, Japan for library preparation and total mRNA NGS. Sample initial quality control was confirmed by Macrogen and the samples proceeded to the next steps. The TruSeq stranded mRNA library preparation kit was used. Samples were sequenced in paired ends at a 101-bp read length using the NovaSeq sequencing platform. After sequencing, results were again checked and the quality measured by Macrogen, Japan. Approximately\u0026thinsp;\u0026gt;\u0026thinsp;4 Gbp of data for each sample with a GC content\u0026thinsp;\u0026gt;\u0026thinsp;30% and Q30\u0026thinsp;\u0026gt;\u0026thinsp;94% reads were obtained for each sample. MD5 values were checked for data integrity upon download.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuality control and data analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuality control\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter sequencing, initial quality control was performed by Macrogen, Japan. Quality assessment was conducted using FastQC and FastTP, considering only good quality reads for further analysis. A quality Phred score cutoff of 25 was applied for subsequent analysis steps. Trim Galore was utilized for adapter trimming and removal of low-quality reads. The version used was Trim Galore 0.6.10 with Cutadapt version 4.1, running on Python 3.8.5. The adapter sequence 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR) was removed. Paired-end trimming mode was employed, resulting in approximately 33% of reads containing adapters, with all reads passing filters.\u003c/p\u003e\u003cp\u003e\u003cb\u003eData analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSTAR alignments were performed to align the paired-end clean reads with the reference genome of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7. We utilized STAR version 2.7.10b, for this purpose. BAM files were generated, and Samtools was employed to create a BAM index file for subsequent analysis. Command-lines are available in the supplementary data (Supplementary data 1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of RNA editing-related genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRaw reads were further analyzed by using iDEP [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Raw read counts were uploaded to the iDEP interface, and the reference genome of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e was selected or automatically matched to the best matching \u003cem\u003ePlasmodium falciparum\u003c/em\u003e genome. Dseq1 and Dseq2 analyses were performed via the iDEP interface. The raw read data were also analyzed using iDEP v1.0 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The data were initially filtered to remove reads below 0.5 CPM in at least one sample. Then, the data were transformed using EdgeR with a pseudocount of 4, and missing values were imputed using gene median. Of the 5767 genes in 12 samples (three replicates for each), 5397 passed the filter. These 5397 genes were converted to Ensembl gene IDs. Transformed group expressions were further analyzed by using iDEP1.1 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Three biological replicates for each condition were included in the analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA variation detection by REDItools1, Denovo approach\u003c/b\u003e\u003c/p\u003e\u003cp\u003eREDItools1, using a denovo approach, primarily compares RNA sequencing data from different experimental conditions. Reference genomic and mRNA sequences are not essential for this analysis. However, we utilized the reference genomic sequence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7 for the better analysis. The parameters used include a minimum per base coverage of 10, a minimum per base quality of 30 (specified by the -q option), a significance value of 0.05, and a minimum variation frequency of 0.10.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA editing detection by REDItools2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe REDItools2 approach involves comparing mRNA sequencing data with either the reference genome or a genomic sequence database. In this study, we utilized the reference genome of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7. Different RNA editing events were counted in a stage-specific manner using the REDItools output tables, with three biological replicates analyzed. Grep commands were employed to identify editing locations with reference nucleotide variation data, excluding SNPs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExploring pathways associated with RNA editing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGene Ontology (GO) pathways were analyzed by using iDEP resources [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Molecular pathways were examined among the three pathways assessed. We observed that RNA editing-related molecular pathways were predominantly downregulated at the 40-h stage compared to the other parasitic stages. The interrelated pathway was also analyzed by using iDEP resources [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The degree of relationship between different RNA modification pathways and their expression was assessed.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eFlow chart of the study\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA visual representation of the processes and workflow involved in this study are presented in the flow chart (Flow chart 1).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample preparation and data analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePCA (principal component analysis) indicated that samples from the same time course clustered together (Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Clustering of the heat map of 2100 genes also indicated that the sample preparation was suitable for further analysis of the RNA sequence data (Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA variation detection by REDItools1, Denovo approach\u003c/b\u003e\u003c/p\u003e\u003cp\u003eREDItools1, Denovo approach provides the variation among RNA sequence data with or without comparing to a reference sequence. Here, we compared three replicates of RNA sequencing data with the reference \u003cem\u003ePlasmodium\u003c/em\u003e genome. A-to-G, G-to-A, T-to-A, T-to-C, and C-to-T variation was detected at a much higher level than the other variations by using REDItools1; denovo approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By using the REDItools 1 denovo approach, we found that the A-to-G and G-to-A conversions were almost similar. A-to-G events at 16 h, 24 h, 32 h, and 40 h were found at 84497, 85847, 87897, and 108198 sites, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). G-to-A events at 16 h, 24 h, 32 h, and 40 h were found at 74988, 76731, 81508, and 88817 sites, respectively. C-to-T events at 16 h, 24 h, 32 h, and 40 h were found at 44952, 41313, 40223, and 47903 sites, respectively. T-to-C variations at 16 h, 24 h, 32 h, and 40 h were found at 67737, 63551, 62792, and 71170 sites, respectively. The proportions of A-to-G, G-to-A, C-to-T, and T-to-A RNA variations were 18%, 16%, 8%, and 11%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In this approach, the pattern of modification was almost the same except for the number of base modifications or variations (Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA editing detection by REDItools2\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe identified the conserved RNA editing events in \u003cem\u003ePlasmodium\u003c/em\u003e and detected how the process continues during the \u003cem\u003ePlasmodium\u003c/em\u003e life cycle. The reference data of RNA variation were used in grep command with our data tables to identify the conserved RNA-editing types, locations, and numbers. In analyses using REDItools 2, the frequency of G-to-A editing was found to be higher than that of A-to-G editing. This finding indicates that the amination reaction is more frequent than the deamination-type RNA editing. C-to-T was higher than T-to-C. In this case, deamination-type RNA editing was more frequent than the amination-type RNA editing. A-to-G editing events at 16 h, 24 h, 32 h, and 40 h were found 60, 66, 71, and 58 sites, respectively. G-to-A editing events at 16 h, 24 h, 32 h, and 40 h were found at 217, 236, 259, and 213 sites, respectively. C-to-T editing events at 16 h, 24 h, 32 h, and 40 h were found at 194, 220, 246, and 200 sites, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). T-to-C editing events at 16 h, 24 h, 32 h, and 40 h were found at 55, 64, 66, and 65 sites, respectively. In our study using REDItools2 and strict screening, we detected various types of editing, including C-to-G, T-to-G, A-to-T, A-to-G, G-to-A, T-to-C, and A-to-C transitions. Among these, deamination-type RNA editing, A-to-G and C-to-T, was found at frequencies of 5% and 17%, respectively. G-to-A and T-to-C occurred at frequencies of 18% and 5%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and represent rare events of amination-type RNA editing in animals and plants. Almost all types of RNA editing started in early developmental, that is, with ring-stage parasites, and gradually increased with for trophozoites and then declined significantly for schizont-stage parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From 16 h to 32 h, there was a significant increase in RNA editing events (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Conversely, from 32 h to 40 h, there was a significant decrease in RNA editing events. These findings indicate that RNA editing starts at the early ring stages of the parasite and gradually decreases as the parasite reaches maturity. In the REDItools2 analyses, samples from the batch 1, batch 2, and batch 3 replicates exhibited the same pattern and extent of RNA editing (Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The genome-wide editing event patterns were almost identical across all parasitic stages in the three biological replicates (Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of RNA editing-related genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eADA2, ADA, Cytidin Deaminase, PPR, cytidin and deoxycytidylate deaminase, and PPR1 have previously been implicated in RNA editing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These genes are homologues RNA-editing genes in other species including higher animals and plants. The expression patterns of these genes are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. ADA2 and ADA were found in almost all parasitic stages but their expression did not correlate with the editing pattern, which was higher in the mature parasitic stages. Cytidine deaminase expression was found to be higher in the ring parasitic stages but gradually decreased in the late stages of the parasite. The expression of cytidine deaminase was negatively correlated with the editing pattern in \u003cem\u003ePlasmodium\u003c/em\u003e. Cytidine and deoxycytidylate deaminase also positively correlated, to some extent, with RNA editing, but to a lesser extent than cytidine deaminase. The expression of cytidine and deoxycytidylate deaminase slightly increased in the ring stages and decreased in the late parasitic stages. In addition, the expression of homologues of PPR and PPR1, important enzyme families in plants, were negatively correlated with the editing pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, the expression of PPR and PPR1, proteins responsible for RNA binding and modification, decreased in the ring stage and increased in the later parasitic stages.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of Zinc fingure genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSome Zinc fingure genes have been reported to be involved in RNA editing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We studied the genes PF3D7_1209300 and PF3D7_1009400, which belong to the C2H2 subfamily; PF3D7_1419900, which belongs to the CCCH subfamily; PF3D7_0627300, PF3D7_1422500, PF3D7_1235300, PF3D7_0512300, and PF3D7_1014600, which belong to an RFP subfamily; PF3D7_1008100, PF3D7_1433400, PF3D7_0629700, and PF3D7_1322100, which belongs to the PHD subfamily; and PF3D7_0314700 and PF3D7_1221000, which belongs to another RFP subfamily. High expression of PF3D7_1009400, PF3D7_0627300, PF3D7_1433400; PF3D7_0629700, and PF3D7_1235300 was found in almost all stages. The expression of PF3D7_1209300, PF3D7_1422500, and PF3D7_1221000 started in the ring stage and gradually increased in the later parasitic stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, PF3D7_1419900, PF3D7_1235300, PF3D7_0512300, and PF3D7_1322100 expression was high at the ring stage and decreased gradually during the later stages of parasitic development (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Expression of the other genes was almost similar in the ring, trophozoites, and schizont stages of the parasites.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathways related to RNA editing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the comparisons between 40 h-16 h, 32 h-16 h, and 24 h-16 h, 1930, 1655, and 807 genes were downregulated, respectively. The downregulated pathways include RNA binding, nucleic acid binding, and catalytic activity acting on RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The RNA binding pathway is particularly important for RNA editing. The 50 most upregulated gene numbers were increased at the 40-h parasite stage. However, we observed a gradual downregulation of the RNA binding pathway up to the 40-h stage of the parasite (Supplemen Additional file 1 tary Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In the comparison between the 24 h and 16 h stages, pathways probably related to RNA editing were not found to be downregulated in the 24 h stage parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Analyses of the relationships among the enriched Gene Ontology (GO) molecular function terms, including RNA binding, nucleic acid binding, and catalytic activity acting on RNA pathways, indicated that these downregulated pathways are related to RNA-editing factors. In the analyses of the network of interrelated pathways, we observed that in the 40 h-32 h comparison, the most downregulated pathways were nucleic acid binding and catalytic activity acting on RNA pathways. Moreover, the catalytic activity acting on RNA pathway shares genes with the RNA methyl transferase activity pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the 40 h-16 h comparison, the highly downregulated pathways included RNA binding pathways containing 292 genes, nucleic acid binding pathways containing 383 genes, and catalytic activity acting on RNA pathways containing 147 genes. In the 32 h-16 h comparison, the moderately downregulated pathways consisted of RNA binding pathways containing 195 genes, nucleic acid binding pathways containing 269 genes, and SnoRNA binding pathways containing only 13 genes. For the 24 h-16 h parasite stage comparison, the highly downregulated pathways were host cell surface binding and cell adhesion molecule binding pathways. Interestingly, pathways related to RNA editing showed no downregulation in the 24 h ring-stage parasites when compared to the 16 h stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, in the 40 h-16 h comparison, the most downregulated pathway at 40 h was the RNA binding pathway. In the 40 h-16 h, 40 h-24 h, and 40 h-32 h comparisons, 821 genes were commonly downregulated in all groups and downregulated in the 40-h samples. However, in the 24 h-16 h, 32 h-24 h, and 40 h-32 h stage comparisons, only 61 genes were commonly downregulated at the 24 h, 32 h, and 40 h stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Of 20 genes tested, those most significantly affected were found to be downregulated at the 40-h stage (Additional file 1 Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, the RNA binding pathway shared common genes with the heterocyclic compound binding, nucleic acid binding, and organic cyclic compound binding pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRNA editing is an important post-transcriptional phenomenon in animal, plants, and microorganisms that increase proteome and transcript diversity. Here, we investigated genome-wide RNA editing that contributed to transcriptional variation.\u003c/p\u003e\u003cp\u003eWhen we utilized the REDItools Denovo approach, we found a high number of SNVs. Previous reports have indicated that the highest numbers of variations in \u003cem\u003ePlasmodium\u003c/em\u003e involve A-to-G, T-to-C, and A-to-T substitutions. Among these, A-to-G and C-to-T SNVs comprise approximately 28% and 4%, respectively [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the Denovo approach, we found that A-to-G and G-to-A conversions occurred in almost similar proportions. This observation suggests that both deamination and amination types of conversions occur in \u003cem\u003ePlasmodium\u003c/em\u003e at similar rates.\u003c/p\u003e\u003cp\u003eWe then employed the REDItool2 approach and screened out the SNPs and selected the conserved RNA editing sites by comparing them with previously identified nucleotide variation sites genome-wide [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Next, we screened out the SNPs and selected only the conserved RNA editing sites between two laboratory-adapted \u003cem\u003ePlasmodium\u003c/em\u003e species. We used the grep command and observed a significant reduction in the number of RNA editing events, decreasing from thousands to hundreds. The most common type of RNA editing events were A-to-T, G-to-A, T-to-A, and C-to-T. We found a higher level of C-to-T type editing, and the enzyme responsible for this conversion is cytidine deaminase. Of note, we used highly synchronized \u003cem\u003ePlasmodium\u003c/em\u003e so we could observe stage-specific events. A-to-G and C-to-T RNA editing usually happen in humans and animals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In plants, T-to-C type editing occurs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our study, the editing types in \u003cem\u003ePlasmodium\u003c/em\u003e are more diversified than those reported for other species.\u003c/p\u003e\u003cp\u003eWe observed a significant difference in the total number of RNA editing events across the developmental stages of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7 parasites. Such variation may help parasites acclimate to the temperature fluctuations in the host. These types of significant changes were observed for only 8-h time intervals, indicating that the RNA editing starts at the early ring stage of the parasites, increases during the trophozoite stage, and gradually decreases when the parasites reach the schizont stage. Genome-wide editing events were almost identical in all parasitic stages across the three biological replicates. Similar developmental stage-specific phenomena have also been observed in \u003cem\u003eXenopus\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, we detected a significant level of RNA editing occurring within a short 8-h period. Editing begins during the early ring parasitic stages, increases up to the 32 h stage, and then decreases significantly. These findings suggest that stage-specific RNA modification is an important phenomenon in this parasite. In our previous study in HEK-293 cells, we observed a considerable level of RNA editing occurring with engineered human deaminase enzymes within a short period [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It has recently been reported that Adenosine Deaminase Acting on RNA (ADARs) have evolved in birds to function optimally at 40\u0026deg;C, targeting different groups of temperature-sensitive RNAs [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. During the early ring stages and the late schizonts stage, prior to the organism encountering the higher core body temperature of its host, the recognition of \u003cem\u003ePlasmodium\u003c/em\u003e RNA by ADARs complementary factors may be altered. Temperature may affect the expression of the RNA-editing factors and the structure of the RNA [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Hence, RNA editing may play a vital role in the rapid and intermittent adaptation of malaria organisms to higher host core body temperatures.\u003c/p\u003e\u003cp\u003eIn most previous reports, RNA editing has been found to be linked to the expression of RNA-editing enzymes and tissue specificity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The editing enzymes are mostly deaminases. In plants, PPR [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and zinc finger proteins are also involved in RNA editing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The ADA gene is essential for malaria parasite survival and adenosine modification in \u003cem\u003ePlasmodium\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Adenosine conversion by ADA has been reported \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. High levels of cytidine deamination are observed with higher expression of cytidine deaminase in the ring stage of parasites, and animal and plant deaminase gene expression is usually proportional to RNA editing [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Hence, in malaria parasites, ADA and other groups of genes may be involved in RNA modification, RNA editing, and parasite adaptation. Of note, the substrate recognition of ADA in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e differs significantly [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] .\u003c/p\u003e\u003cp\u003eMore than 30 Zinc-finger family protein are present in \u003cem\u003ePlasmodium\u003c/em\u003e. These genes are responsible for nucleic acid binding and RNA modification [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In plants, representatives of the zinc-finger family protein are involved in RNA binding, RNA modification, and RNA editing processes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In \u003cem\u003ePlasmodium\u003c/em\u003e, the presence of ADA deaminase enzymes, PPR proteins, and cytidine deaminase has been reported [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Other genes responsible for RNA modification in \u003cem\u003ePlasmodium\u003c/em\u003e need to be identified, as genome-wide RNA editing has been observed throughout the parasitic developmental stages. Here, we found that the expression of ADA is consistent throughout the parasitic developmental stages. Another possibility is that there are complementary RNA editing factors related to \u003cem\u003ePlasmodium\u003c/em\u003e RNA editing that have not yet been reported. The RNA editing system of \u003cem\u003ePlasmodium\u003c/em\u003e might resemble the plant RNA editing system, which comprises a combination of different editosome complexes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt was previously assumed that the malaria organism undergoes numerous transcriptional errors during its multiplication and adaptation to various environments. However, the organism is well-equipped to maintain its high AT-rich genome, comprising approximately 70%, with its transcriptional machinery [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. It can tightly control every step of gene regulation, chromatin structure [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] transcription [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and translation, [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] all of which are essential for its adaptation and survival in diverse host environments.\u003c/p\u003e\u003cp\u003eIn this study, we identified a correlation between the number of RNA editing events and the pathways related to RNA editing. In the 40-h samples, most of the pathways related to RNA editing were highly downregulated and the number of RNA editing events decreased significantly. In the 32-h stage parasites, the major pathway related to RNA editing and the catalytic activity acting on RNA were not downregulated. Furthermore, other pathways related to RNA editing were less downregulated and contained fewer genes compared to the 40-h trophozoite-stage parasites. Based on these findings, we conclude that these pathways and genes are likely to be related to RNA editing as a developmental phenomenon in \u003cem\u003ePlasmodium\u003c/em\u003e parasites. In the 40 h to 16 h stages, the number of RNA editing events was lower than in the other two stages, suggesting that the pathway involving catalytic activity acting on RNA may contain genes responsible for RNA editing.\u003c/p\u003e\u003cp\u003eRNA-editing factors bind in the editing sites of the RNA. These editing sites are mainly defined based on the binding activity of the enzyme or enzyme complex responsible for the RNA editing [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The substrate recognition groove of the ADA enzyme in \u003cem\u003ePlasmodium\u003c/em\u003e is diversified [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003ePlasmodium\u003c/em\u003e ADA not only binds to adenosine (A) but also has a binding pocket for inosine (I), which acts as a homologue of guanosine (G) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This indicates that ADA may be responsible for both deamination and amination. Therefore, it is possible that both deaminase and aminase types of enzyme systems, along with other editosome complexes, may be present in \u003cem\u003ePlasmodium\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIt has been reported that RNA editing is not conserved even at the species level [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This suggest that it may be an adaptive mechanism of the organism to its environment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Our study revealed that genome-wide RNA editing contributes to transcript diversity in different developmental stages. The life cycle of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is 48 h, during which time the enzymatic activity of the various components of the RNA modification machinery encoded in the parasite genome may be critical. Recently, it has been reported that extensive temperature-responsive reversible RNA editing phenomena occur within hours in octopus neural cells, leading to functional diversity in motor proteins such as kinesin and synaptotagmin [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, a stage-specific environmental stress-responsive RNA editing strategy has been identified in fungi [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In ducks and hummingbirds, elevated core body temperature shapes their deaminase system to recognize temperature-sensitive RNA, thus enabling them to adapt to their environment [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Tissue-specific temperature-dependent RNA editing has also been reported in zebrafish [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. RNA editing might, therefore, be an important mechanism of acclimatization in the malarial pathogen, similar to these other organisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Accordingly, we believe that RNA editing plays a vital role in the biology and adaptation of the malaria parasite. It has been reported that single nucleotide alterations in \u003cem\u003ePlasmodium\u003c/em\u003e spp. can lead to antimalarial drug resistance [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Temperature plays a critical role in shaping RNA and RNA editing [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Malaria parasites are exposed to different temperature within the host and various systems. By utilizing the RNA editing machinery, the organism may shape it transcriptomes. Malaria organisms have adapted to a wide range of hosts and to different environmental pressures. RNA editing may play a vital role in adaptation, especially to the transient and intermittent febrile temperatures of the host.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study represents the first comprehensive investigation of genome-wide, stage-specific RNA editing in \u003cem\u003ePlasmodium\u003c/em\u003e nuclear transcripts. We identified conserved RNA editing sites in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7, shedding light on the presence of RNA editing events throughout the \u003cem\u003ePlasmodium\u003c/em\u003e developmental cycle. The factors involved and the targeted RNA require further investigation across \u003cem\u003ePlasmodium\u003c/em\u003e species. Understanding heat stress adaptation, multiplication, and the factors related to RNA editing will be crucial for controlling this dangerous, drug-resistant pathogen. Our findings reveal RNA editing as a developmental phenomenon, with genome-wide conserved events detected at 8-h intervals. We observed a higher number of editing events during the ring to trophozoite developmental stages within human red blood cells, despite the short time intervals between the different developmental stages in malaria parasites. It is challenging to differentiate RNA editing differences due to these short intervals. However, it is important to note that the pipeline used in this study may have excluded certain RNA editing sites from the representative results due to limitations of the grep command. Further studies employing single-cell genome-wide transcriptomic sequencing and single-cell long-read transcriptomic sequencing will provide more detailed and conclusive insights into RNA editing in \u003cem\u003ePlasmodium\u003c/em\u003e. More invasive and focused approaches, such as gene-specific and long-read sequencing, are needed to investigate RNA editing and its consequences in malaria parasites. A detailed understanding of such adaptive strategies may provide fundamental insights for drug and vaccine development targeting this deadly pathogen.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS Fellows (22F21389), and\u0026nbsp;Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan, and by a Livestock Promotional Subsidy from the Japan Racing Association.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Conceptualization,\u0026nbsp;M.T.A.A. and U.Q.; methodology,\u0026nbsp;M.T.A.A.; software,\u0026nbsp;M.T.A.A. and T.S.; validation,\u0026nbsp;M.T.A.A.; formal analysis,\u0026nbsp;M.T.A.A.; investigation,\u0026nbsp;M.T.A.A.; resources,\u0026nbsp;K.K.; data curation,\u0026nbsp;M.T.A.A.; writing—original draft preparation,\u0026nbsp;M.T.A.A.\u0026nbsp;and\u0026nbsp;U.Q.; writing—review and editing,\u0026nbsp;M.T.A.A.,\u0026nbsp;K.K.\u0026nbsp;and\u0026nbsp;U.Q.; supervision,\u0026nbsp;K.K.; project administration,\u0026nbsp;K.K.; and funding acquisition,\u0026nbsp;K.K. and M.T.A.A.\u0026nbsp;\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization (WHO). 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Nat Commun. 2022;13:5746.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRieder LE, Savva YA, Reyna MA, Chang YJ, Dorsky JS, Rezaei A, et al. Dynamic response of RNA editing to temperature in drosophila. BMC Biol. 2015;13.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DNA, Gene modification, mutation, Time-course, Variation","lastPublishedDoi":"10.21203/rs.3.rs-7233611/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7233611/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eRNA editing is an important post-transcriptional modification of RNA. While transcriptional variation is a well-studied phenomenon in \u003cem\u003ePlasmodium\u003c/em\u003e, post-transcriptional modifications, such as RNA editing, have not received as much scrutiny.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eWe detected genome-wide RNA editing in a developmental stage-specific manner at 16 h, 24 h, 32 h, and 40 h of tightly synchronized \u003cem\u003eP. falciparum\u003c/em\u003e 3D7 by using the RNA editing computational approaches REDItools1-Denovo and REDItools2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e REDItools1, the Denovo approach revealed extensive A-to-G, G-to-A, and T-to-C type variations in almost all stages. With the REDItools2 approach and screening, almost all editing events were observed in time-specific parasitic stages. G-to-A and C-to-T events were found at much higher levels. We observed significant differences in stage-specific RNA editing at 8-h intervals. RNA editing was observed at the early ring stages of the parasites, gradually increasing and then decreasing leading up to the 40-hour mature stage. Adenosine deaminase expression did not correlate with the editing level in a time-dependent manner. However, the expression of cytidine deaminase was found to be higher in the ring stages of the parasites, gradually decreasing in the later stages. The expression of other RNA editing-related factors was similar in all developmental stages. Pathways associated with RNA editing were found to be downregulated at the 40 h stage, including RNA binding, nucleic acid binding, and catalytic activity acting on RNA pathways. These findings suggest the presence of robust RNA editing machinery in \u003cem\u003ePlasmodium\u003c/em\u003e, facilitating rapid base conversions within a short timeframe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Our findings will be helpful in identifying the RNA editing machinery, which could serve as a potential tool for antimalarial drug discovery and malaria control in the future.\u003c/p\u003e","manuscriptTitle":"Genome-wide detection of conserved stage-specific RNA editing events for nuclear genes in the Plasmodium falciparum 3D7 malaria parasite","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 14:01:52","doi":"10.21203/rs.3.rs-7233611/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"832a3fa5-1232-4f31-8e43-faa6b5693669","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T12:09:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 14:01:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7233611","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7233611","identity":"rs-7233611","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}