RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii

RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii | 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 Article RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii Abhijit Deshmukh, Kalyani Aswale This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3875304/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The protozoan parasite Toxoplasma gondii is thought to rely on RNA processing to accomplish the differential gene expression needed during life cycle stage transitions. Here, we show how RNA capping, the first major pre-mRNA processing event, safeguards transcript homeostasis in Toxoplasma . A functional RNA capping system of Toxoplasma consists of separate RNA triphosphatase, guanylyltransferase, and guanine-N7-methyltransferase enzymes, which together add 5’ 7-methylguanosine (m 7 G) cap to RNA. The in vitro generated capped RNAs bind to the Toxoplasma translation initiator factor, eIF4E, and are translated to protein in the transfected parasites. Biochemical and genetic characterization demonstrates that among three capping enzymes, triphosphatase (TgRT) is unique and a member of the tunnel family of metal-dependent phosphohydrolases, structurally and mechanistically unrelated to the human cysteine-phosphatase-type RNA triphosphatase. We show that TgRT is essential for pre-mRNA capping and parasite growth through inducible conditional knockdown. TgRT perturbation leads to global diminished m 7 G-capped transcripts, as demonstrated by cap-seq, which resulted in the complete arrest of parasite replication in the culture and the mouse host, protecting them from lethal infection. Overall, this study shows the essential role of TgRT-mediated mRNA capping for parasite survival, thereby presenting RNA triphosphatase as an attractive target for Toxoplasma infection. Biological sciences/Microbiology/Parasitology/Parasite biology Biological sciences/Biochemistry/RNA apicomplexan parasite Toxoplasma gondii mRNA capping RNA triphosphatase Cap sequencing auxin-inducible degron Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Toxoplasma gondii is an extremely successful obligate intracellular apicomplexan parasite that causes lifelong chronic infections in almost all warm-blooded animals, including humans, and severe disease in fetuses and immunocompromised individuals 1 . An estimated one-third of the global human population is chronically infected with T . gondii 2 . Following strong host immune response during the acute infection, the rapidly multiplying tachyzoites differentiate into a slow-growing encysted bradyzoite and develop chronic infection 3 . A fast conversion of bradyzoites into tachyzoites leads to severe mortality in immunosuppressed individuals if not treated. The standard treatment drugs for toxoplasmosis are few and have severe side effects on long-term use, target the tachyzoite stage of the parasite, and are ineffective against encysted bradyzoite in the tissues, necessitating the identification of new drug targets and therapeutics 4 . The complex multi-host life cycle of Toxoplasma involves changes between life stages with distinct morphologies, metabolisms, and reproductive niches. Parasite utilizes an intricate transcriptional, post-transcriptional, and epigenetic network to regulate gene expression required to thrive in these rapidly changing environments and hosts 5 , 6 . The growing evidence suggests that RNA processing, especially co-transcriptional and post-transcriptional mechanisms, is crucial for regulating gene expression during life cycle stage transitions and environmental adoption; however, the underlying mechanism is yet to be discovered in Apicomplexa parasites. In eukaryotes, 5’ mRNA capping is the first and one of the essential co-transcriptional modifications to produce mature mRNA 7 . The 5' cap is essential throughout the life cycle of the mRNA in coordinating various functional processes accomplished by interacting with the cap-biding complex (CBC). The nuclear CBC facilitates splicing, polyadenylation, and export into the cytoplasm, whereas the cytoplasmic CBC containing eukaryotic initiation factor 4F (eIF4F) binds to the cap and recruits the 40S ribosomal subunit to initiate translation 8 . All eukaryotic mRNA contains a 5' cap structure of 7-methylguanosine (m 7 G) linked via 5′ to 5′ triphosphate bridge to the first transcribed nucleotide (m 7 GpppN). The m7GpppN cap structure is formed by a series of three enzymatic steps: (i) hydrolysis of the 5′ triphosphate (pppN) end of the nascent RNA to a diphosphate (ppN) by RNA triphosphatase ; (ii) transfer of a guanine monophosphate (GMP) nucleotide to the 5′-diphosphate RNA (GpppN-guanosine cap) by RNA guanylyltransferase; and (iii) addition of a methyl group to the N7 amine of the guanine cap (m7GpppN) by guanine-N7 methyltransferase. These three catalytic activities, collectively called ‘capping enzymes,' are encoded by separate genes in yeast, whereas in metazoans, the first two capping steps are catalyzed by a single enzyme consisting of two functional domains, the N-terminal triphosphatase and the C-terminal guanylyltransferase 8 – 11 . The structures and mechanisms of the mammalian and fungal capping enzymes have been well elucidated; however, studies on the capping enzymes of protozoa 12 – 15 , except Trypanosoma 16 , 17 , are mostly limited to their biochemical characterization. While guanylyltransferase (GTase) and methyltransferase (guanine-N7 MTase) are conserved, RNA triphosphatase (TPase) has a distinct structure and mechanism of action among eukaryotes. Consequently, RNA triphosphatases (TPase) are classified into two families: i) the divalent cation-dependent TPase of triphosphate tunnel metalloenzyme (TTM) family found in fungi 18 – 20 and protozoa 12 – 16 and ii) the divalent cation-independent TPase of metazoans and plants of cysteine-phosphatase superfamily found in metazoan and plants 21 – 25 . The TTM-type TPases are essential for growth in fungi 23 , 26 , 27 and Trypanosoma brucei protozoan parasite 17 , and as a result, RNA triphosphatases are considered potential drug targets for these infections. The TPase protein essentiality studies are carried out only in tissue culture; however, its essentiality has never been tested for pathogen survival in the host. Additionally, the impact of a lack of capping in the TPase-deficient pathogens on genome-wide M7G-capped RNA abundance, mRNA expression, turnover, and stability has never been studied. Here, we report a detailed characterization of Toxoplasma capping enzymes and investigate the consequences of RNA triphosphatase depletion on transcript homeostasis and parasite survival. Toxoplasma has a functional three-component capping system consisting of separate TTM-type RNA triphosphatase (TgRT), guanylyltransferase, and guanine-N7 methyltransferase. The m 7 G RNA generated using capping enzymes bind to the translation initiator factor, TgeIF4E, and is successfully translated into protein in the parasite. We used an auxin-inducible degron method to generate TgRT conditional knockdown parasites and show that rapid depletion of TgRT leads to downregulation of global m7G-capped transcripts of essential genes, which resulted in the death of the parasite in the culture and in the mouse host. Lastly, we predicted the structure of TgRT, which will enable us to develop an effective inhibitor with minimal or no impact on structurally similar proteins in the host. Materials and Methods Parasite culture T. gondii tachyzoites of RH and ME49 strains were maintained in human foreskin fibroblast cells (HFFs, ATCC) in DMEM containing 10% foetal bovine serum, 10 µg/ml gentamicin, 1% penicillin-streptomycin, and 2 mM L-glutamine at 37°C and 5% CO2. Tachyzoite to bradyzoite stage differentiation was carried out by incubating the ME49 tachyzoites in bradyzoite induction medium (RPMI pH 8.2) at 37°C for 5 days without CO 2 28 . Cloning, expression and purification of TgRT, TgGT, TgGMT, and TgeIF4E The E. coli codon-optimised ORF of T. gondii RT, GT, GMT 2146 − 3849 (subscript denotes nucleotide coordinates), and eIF4E were synthesised by Life Technologies. All three genes were initially cloned into the pMK-RQ vector (Life Technologies) between Nde I- EcoR I sites, which were further subcloned into the pET-28a or pET-21a (Novagen, USA) (Supplementary Table 1), and recombinant proteins were expressed in E . coli BL21 Rosetta as N-terminal (TgRT, TgGT, and TgGMT) and C-terminal (TgeIF4E) 6-xHis-tag and purified on a nickel-nitrilotriacetic acid-agarose resin column as described previously 29 . Briefly, E . coli transformed with pET28a-TgRT/TgGT/TgGMT/TgeIF4E was grown in 10 ml of Luria-Bertani (LB) medium supplemented with 50 µg/ml kanamycin and 34 µg/ml chloramphenicol overnight at 37 o C. Subsequently, 10 ml of the overnight culture was added to 1000 ml of LB containing the same antibiotics and incubated at 37 o C with vigorous shaking. When the OD 600 reached 0.6, IPTG was added to the culture to a final concentration of 1 mM, and the cells were further incubated at 25 o C for 16 h. The cells were then harvested by centrifugation, and the pellets were resuspended in 50 ml of lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 100 µg/ml lysozyme, 10 mM imidazole, 0.1% Triton X-100, and 0.3 mM PMSF, pH 8.0). After centrifugation, the protein was purified from the supernatant with the use of a Ni 2+ -NTA agarose (Qiagen) and step-eluted with 1-ml aliquots of 20–300 mm imidazole in lysis buffer. The quality of the eluted protein was analysed using SDS-PAGE. The appropriate elutes containing the protein were then dialyzed in 1X PBS and stored at -80°C. TgRT mutants (E366A and E726A) and TgGT mutants (K133A, T134A, D135A, and G136A) were generated using a specific set of primers (Supplementary Table 1) following the Stratagene (210515) site-directed mutagenesis protocol. Recombinant mutant protein purification was performed as for wild-type proteins. Polyclonal antibody raising Mouse polyclonal antibodies to recombinant TgRT, TgGT, TgGMT, and TgeIF4E were generated by primary injection with 30 µg of purified recombinant protein in Freund's complete adjuvant (#F5881, Sigma) followed by four boosts of 20 µg each in Freund's incomplete adjuvant (#F5506, Sigma) at 2-week intervals. Serum was collected after day 60 post immunization. Polyclonal antibodies for Tg- IMC1 29 , SAG1 30 , BAG1 31 , CST1 30 , and Aldolase (ALD) antibodies were used from previous studies. Immunoblotting Filter-purified 2×10 5 parasites were suspended in SDS-PAGE sample buffer and boiled for 10 min before being run on a single lane of a 10% polyacrylamide gel. The gel was then transferred to a 0.2 µm PVDF membrane (BioRad) using a Trans-Blot System (BioRad) for 12 h at 30 V. The PVDF membrane was blocked in 5% (w/v) non-fat milk in PBS for 60 min before being probed with a primary antibody (α-TgRT/TgGT/TgGMT/TgeIF4E-1:500; αHA-1:5,000; α-TgSAG1/TgBAG1/TgALD-1:2,000) in non-fat milk overnight at 4°C. The blot was washed 3x with PBS plus Tween-20 detergent (PBST; 0.1% Tween-20) before probing with either HRP-conjugated α-rabbit or α-mouse-IgG (Invitrogen). The blot was washed and developed using the Clarity Western ECL kit (BioRad) and visualised on a ChemiDoc Imager (Biorad). Immunofluorescence (IF) staining HFFs were grown on glass coverslips until confluent and subsequently infected with T . gondii RH/ME49/TgRT-mAID-HA tachyzoites. The infected HFFs were fixed with methanol-free 4% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS for 15 min at room temperature, and blocked with 5% (w/v) bovine serum albumin (BSA, Sigma) in PBS for 60 min at RT. Primary and secondary antibodies were diluted in 1% (w/v) BSA in PBS. Samples were first incubated with the primary antibody (α-TgRT/TgGT/TgGMT/TgeIF4E-1:100; αHA-1:1,000; αTgIMC1/TgSAG1/TgCST1 -1:2,000) at 25 o C for 60 min, washed 5x with PBS, and then incubated with fluorescent secondary antibodies (1:1000) and 4',6-diamidino-2-phenylindole (DAPI; 300 nM) at 25 o C for 60 min. Secondary antibodies (Invitrogen) were conjugated to either Alexa Fluor (AF) green or red fluorophores and specific to the species of primary antibody used. Samples were then washed 5x with PBS before mounting the coverslip on a glass slide using Vectashield medium (Vector Laboratories). IF staining was visualised using a Leica confocal microscope with a 100X oil immersion objective. Images were processed using las x software (Leica Microsystems). A similar protocol was employed for bradyzoite IF staining. Immuno dot-blot RNA and genomic DNA were isolated from filter-purified parasites using RNeasy kit (Qiagen) and DNeasy Blood and Tissue kit (Qiagen), respectively. Indicated amounts of nucleic acids were spotted onto the Hybond-N + membrane (Sigma) and fixed to the membrane with a UV crosslinker (Bioanalytik Jena). The dried membrane was stained with ethidium bromide or methylene blue stain for 15 min at room temperature to stain the spotted nucleic acids. The membrane was blocked for 30 min in 5% (w/v) skim milk in PBS and washed 3x with PBS. The membrane was first incubated with α-m 7 G antibody (Sigma, 1:1000) at 4 o C for 12 hrs, washed 5x with PBS, and then incubated with HRP-conjugated anti-mouse secondary antibody (Invitrogen) at 25 o C for 60 min. The membrane was washed and developed using the Clarity Western ECL kit and visualised on a ChemiDoc Imager. The Cap sequencing experiment followed a similar procedure to determine the m 7 G level of RNA samples treated with RppH and XrnI from control and IAA-treated parasites. RNA triphosphatase (RT) activity RT activity was assayed 12 , 13 by quantifying the release of 32 Pi from γ 32 P-labelled ATP. Standard reaction mixtures (10 µl) containing 50 mM Tris HCl (pH 7.5), 5 mM DTT, 5 mM MnCl 2 /MgCl 2 /ZnCl 2 /CaCl 2 , 1.5 mM ATP, and TgRT protein as specified were incubated for 30 min at 30°C. The reaction mixtures were applied to a polyethyleneimine-cellulose thin-layer chromatography (TLC) plate, which was developed with 0.75 M potassium phosphate (pH 4.3). The release of 32 Pi from [γ 32 P]ATP was quantitated by scanning the TLC plate with a Phosphor Imager (Typhoon, Cytiva). The 5 mM MnCl 2 and 500 ng TgRT protein were used to test the time-dependent, temperature-dependent, and pH-dependent RNA triphosphatase activity. RNA triphosphatase activity in the presence of different NTPs was assayed using 1.5 mM [γ 32 P]ATP or 1.5 mM [α 32 P]ATP or 1.5 mM [α 32 P]UTP, and 500 ng TgRT protein. Guanylyltransferase (GT) assay GT activity was measured 12 , 13 in reaction mixtures (20 µl) containing 50 mM Tris HCl (pH 8.0), 5 mM DTT, 5 mM MnCl 2 , 1 mM [α 32 P]GTP, and TgGT protein (0.5–4 µg) that were incubated for 30 min at 37°C. The reactions were stopped by adding SDS-PAGE sample buffer and boiling for 10 min. The samples were electrophoresed through a 10% polyacrylamide gel, and the GT-GMP intermediate complex was determined by autoradiography using a phosphor imager. Preparation of m 7 GpppN 32 RNA Template dsDNA (N 32 mer) and RNA variants were prepared as described previously 32 . The dsDNA (Supplementary Table 1) was used to generate pppN 32 RNA using MEGAScript T3 Transcription Kit (Ambion). The pppN 32 RNA was treated with TgRT (as described in the TgRT activity) to obtain ppN 32 RNA, and the ppN 32 RNA was treated with TgGT (as described in the TgGT activity) to obtain GpppN 32 RNA. To obtain m 7 GpppN32 RNA from GpppN 32 RNA, GpppN 32 RNA was incubated with TgGMT protein in a reaction mixture containing capping buffer for 30°C for 3 h. The vaccinia capping enzymes (VCE, NEB) was used in the reaction to convert pppN 32 RNA to m 7 GpppN 32 RNA. The obtained RNA samples were heated at 95 o C for 2 min and purified using an RNA cleanup and concentrator kit (Zymo). Luciferase expression assay The firefly luciferase gene was amplified (from the pmirGLO plasmid, Promega) by PCR using the forward primer (Supplementary Table 1) containing the T3 promoter sequence and the starting sequence from the luciferase gene. The obtained DNA was gel-purified and subjected to IVT using the MEGAScript T3 Transcription Kit (Ambion). The reaction was incubated at 37°C for 3 h, treated with DNaseI at 37°C for 1 h, and IVT luciferase RNA was purified using the RNA cleanup kit. The luciferase RNA (ppp-FLuc RNA) was treated sequentially with TgRT, TgGT, and TgGMT proteins or VCE to obtain m 7 G-FLuc RNA. The Poly(A) tailing of ppp-Fluc RNA-[A] n or m 7 G-FLuc RNA-[A] n was performed using E . coli Poly(A) Polymerase (NEB# M0276). Around 20 µg of each RNA variant was used to transfect 10 7 RH parasites using the Gene Pulser Xcell Total System (Biorad, #1652660). Transfected parasites were immediately transferred to a new flask containing a confluent HFF monolayer. Parasites were harvested 18 hrs post-infection, and luciferase activity was determined using the Luciferase Reporter Assay System (Promega, # E1910). Microscale thermophoresis (MST) assay MST assays were performed with a Monolith NT.115 (Nanotemper). His-tagged TgRT protein was labelled with RED-MALEIMIDE 2nd generation dye according to the protocol provided by Nanotemper. Dilutions of the RNA variants (pppN32, GpppN32, and m 7 GpppN32) in a range of 0.18 nM to 600 nM were prepared in in a buffer containing 50 mM HEPES, pH 7.2, 1 mM EDTA, 1 mM DTT, and 100 mM KCl. The reactions were incubated at RT for 15 min, followed by the loading of reaction mixtures into glass capillaries (Nanotemper). The fluorescence intensity for each reaction was measured by keeping 40% infrared laser power and 60% light-emitting diode. The fluorescence values were analysed using the Affinity Analysis software version 2.3 (Nano Temper) to determine the binding affinity (dissociation constant: KD) between TgeIF4E and the RNA variants used. Generation of auxin-inducible TgRT-mAID-HA transgenic parasites TgRT-mAID-HA transgenic parasites were generated by CRISPR/Cas9-mediated site-specific gene editing using the T . gondii line RH TIR1-3FLAG 33 . A CRIPSR/Cas9 plasmid with a specific guide RNA (gRNA) targeting the 3’ end of RT was generated from the pSAG1::Cas9-U6::sgUPRT plasmid (Addgene, #54467) by a Q5 Hot Start site-directed mutagenesis kit (NEB) and primers (Supplementary Table 1) to induce double-strand DNA breaks and direct the insertion of the PCR fragment. The PCR fragment containing the mAID-3HA tag and the HXGPRT selection was amplified from the vector pTUB1:YFP-mAID-3HA using the primers (Supplementary Table 1) with 40 bp of homology with the 3′ end of RT and Q5 polymerase (NEB) to facilitate direct insertion of the PCR fragment and double homologous recombination. Ten µg each of TgRT-mAID-HA amplicon and pSAG1::Cas9-U6::sgRT were transfected into 10 7 T . gondii RH TIR1-3FLAG parasites by electroporation using the Gene Pulser Xcell Total System (Biorad, #1652660). Transfected parasites were drug-selected using mycophenolic acid (25 µg/ml) and xanthene (50 µg/ml) for two growth cycles before cloning out by serial dilution. Endogenous tagging of TgRT-mAID-HA was verified using sequencing, diagnostic PCR, immunoblotting, and IF staining. The auxin-inducible degradation of TgRT-mAID-HA was tested by culturing these parasites in a medium containing 500 µM indole-3-acetic acid (IAA) (Sigma, I2886), followed by immunoblotting and IF staining using α-HA antibody. Plaque assay HFFs were grown in 6-well plates until confluent and subsequently infected with 100 parasites per well. After 24 h, the medium was removed and the wells were treated with 500 µM IAA or an equivalent volume of MeOH vehicle. Plates remained undisturbed for 5 days before the infected HFFs were fixed with 100% ice-cold MeOH for 20 min and stained with a 1% crystal violet solution for 20 min. Plaques (zone of lysis: white) could be visualised against intact cells (purple). The plaque areas were quantified using ImageJ. Three independent experiments were performed with similar results. For the rescue experiment, a similar plaque assay procedure was followed; however, the IAA-containing medium was replaced with normal parasite medium at the indicated time points. Replication HFFs were grown on glass coverslips in a 6-well plate until confluent and subsequently infected with 10 4 parasites per well. After 12 h, the medium was removed, and the wells were treated with 500 µM IAA or an equivalent volume of MeOH vehicle. 18 hours later, infected HFFs were fixed, permeabilized, stained with α-TgIMC1 (1:2,000) and DAPI, and subjected to IF staining. The number of parasites per vacuole was determined by counting the parasites from 50 random vacuoles. A total of three independent replicates were performed. A similar procedure was followed to count the morphologically defective parasites. Invasion Parasites grown in HFF monolayers were treated with vehicle or IAA for 14 h to deplete TgRT-mAID-3HA protein. Parasites were then harvested, and added to HFF monolayers cultured on coverslips (10 5 parasites/well) for 20 min at 37C in the presence of the vehicle or IAA. Infected HFFs were fixed and stained with rabbit α-TgIMC1 (1:2,000). Following 3x washes with PBS, infected HFFs were permeabilized and stained with mouse α-TgIMC1 (1:2,000) and DAPI. After 3x washes with PBS, infected HFFs were stained with AF-conjugated secondary antibodies (anti-mouse IgG AF 488 and anti-rabbit IgG AF 568) and DAPI. Parasites (green/red) were counted from thirty random fields. The relative efficiency of attachment and invasion of IAA-treated parasites was expressed as a mean percentage of the control treatment from three independent experiments. Egress HFFs were grown on glass coverslips in a 6-well plate and infected with 10 4 parasites per well. After 8 h, the medium was replaced with DMEM containing 500 µM IAA or MeOH vehicle. 24 hours later, parasite egress was triggered by the addition of 3 µM calcium ionophore A23187 to infected HFFs for 2 min. The infected HFFs were fixed, permeabilized, and stained with α-TgGRA2 (1:1000) and α-TgIMC1 (1:2000). A total of 150 vacuoles from each experiment ( n = 3) were examined to count the intact or collapsed vacuoles. Vacuoles containing > 2 parasites were considered intact. Anti-GRA2 (NR-50260) monoclonal antibody was procured from BEI resources. CAP sequencing RT-mAID-HA parasites (5 x 10 7 ) were harvested from infected HFFs grown in DMEM containing 500 µM IAA or MeOH vehicle for 8 h. Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) and subjected to enzymatic treatments, followed by RNA purification using the RNA cleanup kit. First, 5µg RNA was treated with RppH (NEB) in NEB buffer 2 at 37 o C for 1 h, followed by treatment with Xrn1 (NEB) in NEBuffer 3 at 37 o C for 1 h. The treatment of these two enzymes ensured enrichment of 5’m7G and 5’OH RNAs and marked depletion of rRNA. Subsequently, RNA was treated with RppH (NEB) in Thermopol buffer at 37 o C for 1 h to convert m 7 G-RNA to p-RNA, which was confirmed by dot blot using the α-m 7 G antibody. The quality of recovered RNA was assessed using tapestation 4150. The obtained RIN values were < 2, confirming successful rRNA depletion in the RNA samples. The RNA samples were quantified using a Qubit flurometer (Thermofisher #Q33238). The cDNA libraries were prepared using the Truseq Kit (Illumina), followed by the sequencing of paired end reads on the Illumina NovaSeq 6000 platform. The read quality was examined using MultiQC and FastaQC. The reads were mapped onto the Toxoplasma reference genome (GCF_000006565.2) using STAR v2.7.9a, followed by transcript quantification by RSEM. Differential expression analysis (DEA) was performed using DESeq2. The k-means cluster analysis was performed using iDEP.96. The outputs from feature counts (DESeq2 and k-means clustering) were represented using RStudio. Gene ontology analysis was performed using ToxoDB. Mouse infection The auxin-inducible degradation of protein in mice was performed as described previously 34 with some modifications. To test TgRT depletion in vivo, 6-week BALB/c male mice ( n = 4) were injected intraperitoneally (i.p.) with 50 tachyzoites of RH TgRT-mAID-HA (2 groups). From the second day of post-infection (pi), of two groups, one group was given IAA, and the other group was administered an equivalent volume of MeOH vehicle in the drinking water containing 5% sucrose. The IAA was administered in two ways: i) in drinking water (0.5 mg/ml) and ii) by oral gavage (12.5 mg/mL). The total treatment period was 4 days (2 days pi to 5 days pi). On day 5 and 6 pi, 2 mice each were sacrificed by CO 2 asphyxiation, and the peritoneal exudate cells (PECs) were collected and examined by IF staining using α-HA and α-TgIMC1 antibodies to test the TgRT protein depletion. After successful depletion of TgRT-mAID-HA in IAA-administered mice, a long-term (30-day) survival experiment was performed by following a similar experimental procedure. In the 30-day survival experiment, each group had 10 mice (2 groups RH TgRT-mAID-HA); mice were weighed and monitored daily; PECs were collected on days 6 and 12; IAA treatment was given for 15 days in the surviving mice; and survived mice were sacrificed on day 30. Relative weight loss was calculated based on the initial body weight on the day of infection. The overall experimental setup and procedures used are indicated within the relevant figures. Structure predictions The alphafold structure 35 of TgRT (S8F0G9) was obtained from ToxoDB. The disordered region was manually removed and amino acid sequence corresponding to 361–729 aa, which consists of 10β barrel tunnels was used as input sequence for structure prediction using GoogleCollab notebook 36 https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFol d.ipynb#scrollTo = rowN0bVYLe9n. The obtained structure of RT was superimposed with PDB structures of RNA triphosphatase of Saccharomyces cerevisiae (1D8H) 23 , Trypanosoma cruzi (6L7W) 37 and thiamine triphosphatase 38 of Homo sapiens (3VTL) using PyMol (v2.x). Yeast complementation For complementation assays, S . cerevisiae Δcet1 , Δceg1 , Δabd1 , and three genes chromosomal copy deletion mutant strains (Supplementary Table 1) carrying the wild-type copy in a plasmid with URA marker were utilized 39 , 40 . Full-length T . gondii rt , and gt and C-terminus of gmt genes were cloned (Supplementary Table 1) into pYES3/CT vector between Kpn I- EcoR I, BamH I- Xho I, and Kpn I- EcoR I, respectively. The cet , ceg , and abd , yeast mutant stains were transformed with respective plasmid carrying respective T . gondii gene or empty plasmid. Triple deletant mutant strain was transformed with plasmids carrying all three T . gondii genes or empty vector. Transformants were selected on SD –Trp plates with or without 5-Fluoroorotic Acid (5-FOA). Data analyses All data analyses, including graph preparation and statistics, were performed using GraphPad Prism 9. Immunoblot quantification was performed using ImageJ (Version 6). Results T . gondii encodes three separate mRNA capping enzymes To examine the presence of 7-methylguanosine (m 7 G) cap in Toxoplasma , we performed immunofluorescence analysis using an anti-m 7 G antibody in the asexual stages (tachyzoite and bradyzoite) of the parasite. Analysis of intracellular parasites revealed predominant punctate staining for m 7 G at the nuclear periphery and cytoplasm of tachyzoite (Tz) and bradyzoite (Bz) (Fig. 1 A). To detect whether the m 7 G mark is present on RNA, we performed an immuno-dot blot using an anti-m7G antibody on total RNA and genomic DNA (gDNA) extracted from filter-purified tachyzoites. The results demonstrated that the m 7 G mark was exclusively present in RNA (Fig. 1 B). Collectively, these results show that m 7 G cap RNA is a feature of Toxoplasma . To identify capping enzymes, which could add m 7 G structure to RNA, we performed BLASTP homology searches of the Toxoplasma genome ( http://toxodb.org/toxo/ ) using amino acid sequences of yeast Cet1, Ceg1, and Abd1 proteins as queries. The search analysis revealed three separate RNA capping enzymes in Toxoplasma that are similar to yeast. The identified candidate Toxoplasma RNA triphosphatase, guanylyltransferase, and guanine-N7 methyltransferase were named TgRT (TGME49_224650), TgGT (TGME49_305320), and TgGMT (TGME49_272720), respectively (Fig. 1 C). The MEGA analysis was performed to determine how closely the Toxoplasma capping enzymes (TgCEs) are related to the alveolates' and other eukaryotes' capping proteins. Phylogenetic analysis revealed a distinction between two apicomplexan classes, Conoidasida and Aconoidasida , for all three capping proteins. Members of the Sarcocystidae family were found to encode the largest triphosphatase and methyltransferase proteins (Supplementary Fig. 1A-C). The putative TgRT gene encodes a 920-aa polypeptide and has characteristic features of fungal triphosphatases 9 , including two glutamate-containing metal-binding motifs, homologs of β strands that comprise the active site tunnel, and conserved hydrophilic amino acids required for catalysis. (Fig. 1 D, Supplementary Fig. 2). The putative TgGT is a 509-aa protein contains two conserved domains, a nucleotidyl transferase (NTase) domain and a C-terminal oligonucleotide-binding domain (OB) 41 , 42 . The NTase domain has six conserved motifs (I, III, IIIa, IV, V, and VI) 43 (Supplementary Fig. 3) with a lysine-containing KxDG motif I (Fig. 1 D), which comprises the active site of GTP-binding and nucleotidyl transfer 44 , 45 . The putative TgGMT encodes 1283-aa polypeptide with large N-terminal extension and conserved glycine-rich sequence in the SAM-binding motif (Fig. 1 D). Among all three capping enzymes, TgRT and TgGMT are significantly larger than yeast's TPase and guanine-N7 MTase; however, each of the three Toxoplasma capping enzyme orthologue is essential for parasites according to a genome-wide CRISPR screen (Fig. 1 C), suggesting that M 7 G capping of RNA is important and required for parasite fitness. Owing to the sequence similarity between Toxoplasma capping enzymes (TgCEs) and yeast enzymes, we performed yeast complementation to test whether TgCEs function in the cap-synthetic pathway and sustain the growth of yeast cells that lack one or more capping enzymes. We separately cloned the TgRT , TgGT , and TgGMT genes into a yeast 2µ TRP1 pYES3 plasmid, and the function of each of these genes was tested by plasmid shuffle in S . cerevisiae Δcet1 or Δceg1 or Δabd1 cells that contain respective gene on a CEN URA3 plasmid. The mutant strain cannot survive on a medium containing 5-FOA, a drug that selects against the URA3 plasmid unless it is transformed with a second plasmid containing a functional homolog from another source. We found that 2µ TgRT , TgGT , and TgGMT supported the growth of Δcet1 , Δceg1, and Δabd1 cells, respectively (Fig. 1 E-G). Similarly, 2µ TgRT + TgGT + TgGMT supported the growth of triple mutant yeast cells (Fig. 1 H). These results demonstrate that Toxoplasma encodes biologically active capping enzymes. Toxoplasma RNA Triphosphatase TgRT shows metal-dependent triphosphatase activity To gain insight into the expression, localization, and biochemical function of TgRT, full-length His 6 -TgRT protein of ~ 100 kDa was purified (Fig. 2 A) and used to generate specific anti- TgRT antibodies. The expression and localization studies revealed that TgRT is robustly expressed (Fig. 2 B) in both the asexual stages and localized in the nucleus of the tachyzoite and bradyzoite stages (Fig. 2 C), as demonstrated using anti-TgRT antibodies. Further, we tested the triphosphatase activity of TgRT using a detailed biochemical characterization. The recombinant wild type (WT) TgRT catalyzed the release of 32 Pi from [γ- 32 P]ATP in the presence of manganese (Fig. 2 D); however, the ATP hydrolysis was ineffective in the presence of magnesium (Fig. 2 D). The extent of ATP hydrolysis was proportional to TgRT concentration (Fig. 2 E). No ATP hydrolysis was observed without divalent cation (Fig. 2 E). ATP activity was nominal in the presence of calcium or zinc (Fig. 2 F). The optimal ATPase activity of TgRT was detected as early as 5 min and remained stable for 30 min (Fig. 2 G). The enzyme activity of TgRT was similar from 20 o C to 70 o C (Fig. 2 H). TgRT was found to be catalytically active in a wide pH range (5.5–10) with an optimal activity from pH 6.5 to 8.0 (Fig. 2 I). We also tested the specificity for NTP hydrolysis using two different triphosphorylated nucleoside substrates. The rate of release of 32 Pi from [γ- 32 P]ATP was similar to the rate of conversion of [α- 32 P]ATP to [α- 32 P]ADP and [α- 32 P]UTP to [α- 32 P]UDP in a parallel reaction mixture containing the same concentration of TgRT (Fig. 2 J). Two glutamate residues corresponding to the metal binding sites of the tunnel were replaced by alanine (E366A and E726A) (Fig. 1 D and 2 K), and enzyme activities were compared. ATPase activity of the E366A mutant was 50% of the activity of wild-type TgRT (Fig. 2 L), whereas in comparison, E726A showed < 10% activity (Fig. 2 L). Less activity in the mutant proteins was due to a change in the secondary structure, as demonstrated by circular dichroism (Supplementary Fig. 3). Together, these results demonstrated that TgRT belongs to the family of triphosphate tunnel metalloenzymes (TTMs). Characterization of Toxoplasma Guanylyltransferase TgGT The full-length His 6 -TgGT protein of ~ 60 kDa was purified (Fig. 3 A) and used to generate anti-TgGT antibodies. TgGT is robustly expressed in the asexual stages (Fig. 3 B) and localized in the nucleus of the tachyzoite and bradyzoite stages (Fig. 3 C). All known guanylyltransferases accomplish nucleotidyl transfer through a covalent enzyme-(lysyl-N)-GMP intermediate that can be detected by label transfer from [α- 32 P]GTP to the enzyme. To determine the guanylyltransferase activity of TgGT, protein was incubated with [α- 32 P]GTP and a divalent cation, which resulted in the formation of an SDS-stable ~ 60 kDa enzyme-GMP intermediate (Fig. 3 D). TgGT activity requires a divalent cation cofactor, either manganese or magnesium; however, enzyme activity was more effective in the presence of manganese than magnesium (Fig. 3 D). Enzyme-guanylate formation was linear with respect to TgGT concentration (Fig. 3 E). We mutated conserved Lys (essential for GMP interaction during the guanylyltransferase reaction for GTases) 39 , Tyr, Glu, and Gly residues to alanine (K133A, T134A, D135A, and G136A) of TgGT and compared enzyme activities (Fig. 3 F). None of the mutant proteins showed GTase activity (Fig. 3 G); however, after long autoradiographic exposure, a trace of residual activity was observed for T134A (Fig. 3 G). No activity in the TgGT mutant proteins was due to a change in the secondary structure, as demonstrated by circular dichroism (Supplementary Fig. 4). We conclude that the observed guanylyltransferase activity is intrinsic of TgGT. Characterization of Toxoplasma guanine-N7 methyltransferase TgGMT His 6− TgGMT 716 − 1283 (subscript denotes amino acid coordinates) protein of ~ 60 kDa was purified (Fig. 4 A) and used to generate anti-TgGMT antibodies. The expression and localization studies showed TgGMT expressed in the asexual stages (Fig. 4 B) and primarily localized in the nucleus of the tachyzoite and bradyzoite stages (Fig. 4 C). To test the guanine-N7 methyltransferase activity of TgGMT, we first generated 32 mer RNA (pppN 32 RNA) using in vitro transcription and sequentially treated with TgRT to generate ppN 32 , TgGT to generate GpppN 32 , and TgGMT in the presence of methyl donor S-adenosyl methionine (SAM) to generate m 7 GpppN 32 , as shown in Fig. 4 D. The individual reaction was spotted on the membrane and capping of RNA substrate was determined using anti-m 7 G antibody. The immune-blot analysis revealed that TgGMT could successfully add m 7 G to the guanylated RNA (Fig. 4 E). Vaccinia Capping Enzyme (VCE) was used as a positive control to generate m 7 GpppN 32 (Fig. 4 E). The specificity of TgGMT to use SAM was tested using sinefungin, a structural analog of SAM and inhibitor of methyltransferases 46 , 47 . Immunoblot analysis using anti-m 7 G antibody revealed that sinefungin inhibits m 7 GpppN 32 synthesis in a concentration-dependent manner (Fig. 4 F). Together, these results demonstrate that TgCEs are biochemically active and function in a cap-synthetic pathway in Toxoplasma . Cap-dependent translation in vitro The eukaryotic translation initiation factor, eIF4E, binds to the m 7 G cap of mRNA and initiates translation 48 , 49 . Similarly, we wanted to test whether in vitro -generated capped RNA could be recognized by eIF4E-like protein and productively translated into protein in Toxoplasma . BLASTP search using Plasmodium 50 and human 51 eIF4E revealed three eIF4E-like homologues (TGME49_223410, TGME49_315150, and TGME49_312560) in T . gondii genome 52 . Of these three homologue, TGME49_223410 showed highest amino acid similarity, including conserved residues required for m 7 G interaction with Plasmodium (e score: 1e-69) and human protein (e score: 1e-14) (Supplementary Fig. 6). While the eIF4E protein is highly conserved in eukaryotes, TgeIF4E clusters with fungi and other protozoan parasites, and the metazoans form a separate clan (Supplementary Fig. 7). Hence, we name this putative protein TgeIF4E, a 225-aa protein (Fig. 5 A). The full-length TgeIF4E-His 6 protein of ~ 26 kDa was purified (Fig. 5 B) and used to generate specific antibodies. TgeIF4E is robustly expressed in the asexual stages (Fig. 5 B) and, as expected, localized in the cytoplasm of the tachyzoite and bradyzoite stages (Fig. 5 C). To determine the interaction between TgeIF4E with capped or non-capped RNA variants generated using TgCEs, a microscale thermophoresis (MST) assay was performed using fluorescently labeled TgeIF4E. As measured, TgeIF4E showed strong binding affinity (K D =8.01 ± 1 nM) towards m 7 GpppN 32 RNA (Fig. 5 F) and no binding affinity was measured for pppN 32 RNA (Fig. 5 D) and GpppN 32 RNA (Fig. 5 E). These results confirm the m 7 G cap specificity of TgeIF4E. Further, to evaluate m 7 G RNA were utilized by Toxoplasma to promote translation, we generated luciferase transcript variants in vitro with and without 5’-m 7 G cap (using TgCEs) and 3’-poly(A)tails (Fig. 5 G). We found that luciferase activity was detected after transfection of capped and polyadenylated RNA (m 7 G-FLuc-[A] n ), but not after transfected with either only IVT RNA (pppFLuc) or capped RNA (m 7 G-FLuc) or polyadenylated RNA (pppFLuc-[A] n ) 14 (Fig. 5 H). As a positive control, 5’capped and 3’polyadenyaled FLuc RNA was generated using VCE and PolyA polymerase and transfected. However, after comparing the luciferase activity, we found that TgCEs are less effective than VCE (Fig. 5 H). Together, these results demonstrate that in vitro -generated capped RNA using TgCEs can be utilized by Toxoplasma to promote translation in vivo . Depletion of TgRT impairs overall m 7 G levels and arrest parasite replication Toxoplasma RNA triphosphatase is a metalloenzyme unique to the parasite. To understand better the role of this protein in the transcription-associated processes, we endogenously tagged TgRT at the C-terminus with mini auxin-inducible degron (mAID) fused with three copies of HA (3HA) epitope (TgRT-mAID-3HA) in RH strain parasites expressing TIR1, which allows rapid degradation of the TgRT-mAID-HA protein (Fig. 6 A) upon the addition of indole 3-acetic acid (IAA). The resulting TgRT-mAID-HA strain was confirmed by diagnostic PCR (Fig. 6 B). Western blot analysis using anti-HA antibody revealed complete loss of the TgRT-mAID-HA protein in as little as 1 h following addition of 500 µM IAA in the culture medium (Fig. 6 C). Using IF analysis, we also observed no staining for TgRT-mAID-HA in the 1 h IAA treated parasites (Fig. 6 D). Consistent with the role in the mRNA capping, depletion of TgRT for 8 h diminished m 7 G capped RNA in the parasite (Fig. 6 E and 6 F). Initially, we checked the m7G levels of total parasite RNA of control vs treated parasites at 1 h, 4 h, and 8 h; however, we could observe a significant decline in m7G levels after 8 h of TgRT depletion. Hence, we selected an 8 h IAA treatment period for all the further experiments. The effect of TgRT depletion on productive translation was examined by transfecting TgRT-mAID-HA parasites with Luciferase plasmid. As measured, the luciferase activity was decreased after 4 h, and a drastic reduction in the activity was observed after 8 h of RT depletion (Fig. 6 G). Collectively, these results show that depletion of TgRT leads to reduced RNA capping in the parasite. We next examined the effect of TgRT depletion on parasite-specific processes. The complete arrest of parasite replication was observed in the TgRT-depleted parasites tested using a standard parasite counting assay (Fig. 6 H). A significant number of parasites displayed morphological defects upon TgRT depletion (Fig. 6 I). The impact of TgRT depletion on parasite growth was tested using plaque assays. Unlike the parental strain, TgRT-depleted parasites produced no visible plaques (Fig. 6 J-L). To determine whether parasites can recover from a transient loss of TgRT expression, we conducted a plaque assay that used six different IAA treatment regimens (Fig. 6 M). After an initial 24 h of growth, parasites were treated with IAA or vehicle for 1/2/4/8/12/24 h; at this point, the media was replaced with fresh IAA (or vehicle) and incubated for 5 days. The number of plaques formed was similar for 1 h IAA or vehicle-treated parasites (Fig. 6 M and 6 N). Two hours of IAA-treated TgRT-mAID-HA parasites showed a 50% reduction in the number of plaques formed, whereas no plaques were observed when treated for 4 h or 8 h or 12 h, or 24 h IAA (Fig. 6 M and 6 N). These results indicate that parasites could not recover after depletion of capping for > 4 h. The depletion of TgRT significantly decreased the invasion efficiency of the parasite (Fig. 6 O); however, no difference was observed in the ability of parasites to egress upon inducing egress using calcium ionophore (Fig. 6 P). Together, these data provide compelling evidence that TgRT is essential for Toxoplasma viability and proliferation. Depletion of TgRT perturbs gene expression Given that M 7 G RNA capping governs overall RNA metabolism, we reasoned that the loss of parasite viability upon TgRT depletion could be due to a defect in the capped RNA abundance and gene expression. We investigated the consequences of TgRT depletion (the absence of capping) on gene expression using cap sequencing, which was aimed at capturing all the m 7 G capped RNA population to provide a transcriptome-wide profile that will reveal the identity and relative levels of capped RNA. The m 7 G-capped RNAs were enriched using sequential enzyme treatments (Fig. 7 A) on 8 h IAA- or vehicle-treated TgRT-mAID-HA parasites (in biological duplicates). In the first step for the enrichment capped RNA, total RNAs were treated sequentially with RppH (in the presence of NEBbufer2) and Xrn1 to remove rRNA and 5’-monophosphate-ended RNA (Fig. 7 B). Subsequently, RNAs were treated with RppH in the presence of Thermopol buffer to convert M 7 G-capped RNA (decapping) to monophosphate-ended RNA (Fig. 7 C). These decapped RNAs were subjected to library preparation and sequencing, and the obtained transcriptomic data were utilized for differential gene expression analysis (DESeq). The gene expression data from two biological experiments were analyzed using a k-means clustering algorithm. Clustering of 2000 transcripts revealed four clusters (A, B, C, and D) with similar expression profiles in two biological replicates of IAA or vehicle samples (Fig. 7 D). The significantly altered capped transcripts were identified by edgeR (FDR ≤ 0.005; fold change ≥ 1), resulting in 185 and 186 genes whose abundance was decreased and increased, respectively, upon depletion of TgRT (Fig. 7 E). To understand what processes the differentially expressed genes might be involved in, we performed gene ontology (GO) enrichment analyses (Supplementary Table 2). We could identify clear terms for cellular components (CC) and biological processes (BP) for the significantly dysregulated genes. The multiple GO terms were identified for the downregulated genes but were mostly related to ‘DNA packaging’ and ‘cell membrane’. The most significant downregulated genes (Fig. 7 F and 7 H) were histones (H1 like protens, H2A1, H2Ba, H2Bb, H2Ax, H3, and H4) and inner membrane complex proteins (IMC16, IMC17, IMC20, IMC22, IMC26, IMC34, IMC35). Transcripts related to protein phosphorylation (Fig. 7 F and 7 G) were found to be significantly downregulated (Rhoptry kinases-ROPs, aurora kinase, calcium-dependent protein kinase). However, transcripts related to transporter activities (phosphate transporters, MC family transporter) and transferase activity (glucosamine transferases, dehydrogenases, esterases) were found to be significantly upregulated (Fig. 7 F and 7 H). Further analysis revealed glucose (PYK1, ENO1, GAPDH1, ENR, and ACC1) and glutamate metabolism (glutathione synthase, glutamate cysteine ligase) pathways upregulated upon loss of capping (Fig. 7 F and 7 H). Depletion of TgRT protects mice from lethal toxoplasmosis TgRT protein is essential for parasite fitness in tissue culture; however, to test its essentiality in establishing an infection in the host, we performed mouse infection studies. First, we tested whether TgRT-mAID-HA could be depleted in vivo (Fig. 8 A). Mice were infected with 50 tachyzoites of RH TgRT-mAID-HA parasites intraperitoneally and then treated orally with 200 mg/kg/day IAA 34 or vehicle from day 2 to 4 post-infection (Fig. 8 A). On day 5 and 6 pi, the mice were sacrificed and peritoneal exudate cells (PECs) were collected for IF microscopy. Parasites from IAA-treated mice showed depleted TgRT-mAID-HA levels but normal levels of a control protein, TgIMC1 as compared to parasites from the vehicle-treated mice (Fig. 8 B). This experiment confirmed the successful in vivo depletion of TgRT-mAID-HA protein as early as 3 days after IAA treatment. Next, to determine the effect of prolonged depletion of TgRT on parasite survival in vivo, mice were infected with RH TgRT-mAID-HA parasites and then treated with IAA or vehicle orally for 15 days to deplete TgRT (Fig. 8 C). The depletion TgRT was confirmed by IF microscopy in one of the sacrificed mice at 5 day of IAA treatment (data not shown). All mice receiving the vehicle control treatment succumbed to lethal toxoplasmosis by day 11 post-infection (Fig. 8 D). Conversely, IAA treatment ( i.e ., TgRT depletion) rescued all mice from lethal toxoplasmosis, indicating that TgRT is necessary for acute infection in T . gondii (Fig. 8 D). Severe morbidity and complete mortality were seen in the control treatment group compared to the IAA treatment group (Fig. 8 E and 8 D). By day 2 pi, the control group showed weight loss, a sign of illness, and continued to lose up to a quarter of their initial weight by the time of death. The IAA treatment group showed no weight loss. Suppressing TgRT expression during the acute phase of infection effectively blocked replication of T . gondii since discontinuation of IAA treatment following day 15 did not result in morbidity (Fig. 8 E), mortality (Fig. 8 D), or parasite presence (tested by collecting PECs -data not shown) as monitored for an additional 15 days. Overall, these results show the essential role of TgRT for parasite survival and replication in the mouse host. Structural analysis of T . gondii RNA triphosphatase The tunnel family RNA triphosphatases are potential antiinfective targets owing to the complete divergence in structures and mechanisms of the RNA triphosphatases of the unicellular pathogen and the mammalian host. We showed that the mechanism of action of TgRT is different from its host counterpart; however, we have yet to know the structural details of this protein, which can help develop a parasite-specific inhibitor. To gain insight into the structure of TgRT, we first attempted to perform comparative modeling of TgRT with known PDB structures of other RNA triphosphatases; however, we failed to obtain the structure owing to poor (< 40%) sequence homology. Next, we retrieved Alphafold predicted structure of TgRT using the AlphaFold database ( https://alphafold.ebi.ac.uk/entry/S8F0G9 ). The obtained structure showed a moderate to high confidence score (the per-residue model confidence score: 40–95) for the region corresponding to the triphosphate tunnel (361-729aa) and a high predicted error (PAE) for the other regions of TgRT (Supplementary Fig. 8A and 8B). Finally, using a ColabFold, the structure of the triphosphate tunnel region (361-729aa) was predicted (Supplementary Fig. 8C). The predicted structure comprised 10 anti-parallel β barrels (green) with 3 α helices (blue) surrounding the active tunnel site (Fig. 9 A and 9 B). Structural comparison with S . cerevisiae RNA triphosphatase (Cet1) revealed a nearly identical structure (for triphosphatase tunnel) of the two enzymes (Fig. 9 C) with 9 of 15 side chain positions important for Cet1 activity conserved in TgRT (Fig. 9 D). The triphosphatase tunnel structure of TgRT also showed high structural similarity, including active site residues with the recently deciphered crystal structure of Trypanosoma cruzi RNA triphosphatase. (Fig. 9 D and 9 E). Next, we checked whether any host protein has similar structure as of TgRT. Using psi BLAST, we found human thiamine triphosphatase (hTTP) is the only TTM with the available structure that resembles TgRT predicted structure (Fig. 9 F). Structural comparison with hTTP revealed that the metal binding sites are identical between the two enzymes (Fig. 9 D); however, TgRT lacks the C-terminal plug-in helix, which ensures a topologically closed structure and provides substrate specificity only for thiamine triphosphatase (Fig. 9 F). Collectively, these results demonstrate the overall similarity in the structure of TgRT with other TTMs. Although the central tunnel structure of TTM is conserved amongst TgRT and hTTP, the substrate recognition and specificity vary between them, and this raises the possibility of development of an inhibitor that specifically targets the entry of RNA substrate into TgRT with minimal to no effect on the hTTP. Discussion A defining feature of eukaryotic gene expression is the addition of an m 7 G cap to nascent pre-mRNAs shortly after the initiation of synthesis 53 . We aimed to understand the capping process in Toxoplasma to examine parasite-specific variations and to find the contribution of this process to a broader understanding of mRNA metabolism and cellular responses. T . gondii encodes three separate mRNA capping enzymes: triphosphatase, guanylyltransferase, and cap methyltransferase, similar to that of fungi and distinct from the two enzyme capping systems of metazoans and plants. Biochemical characterization of the triphosphatase TgRT firmly categorized it in the family of metal-dependent phosphohydrolases identified in fungi, DNA viruses, and some protozoa. Despite low similarity with other homologs, guanylyltransferase TgGT and the methyltransferase TgGMT are similar in structure and mechanism to the typical guanylyltransferases and cap methyltransferase enzymes found in all eukaryotes. The capping functions of TgCEs were verified biochemically by testing the productive translation of the in vitro generated capped reporter RNA and genetically through complementation of S. cerevisiae strains lacking endogenous enzymes. The depletion of TgRT resulted in global defects in gene expression and complete arrest of parasite replication (tissue culture and mouse host), highlighting the essential role of triphosphatase in the parasite. In eukaryotes, capping is facilitated by direct recruitment of the capping enzymes to the transcription machinery via interactions with the phosphorylated C-terminal domain (CTD) of Rpb1, the largest subunit of RNAP II 54 . In many organisms, guanylyltransferase and methyltransferase interact directly with the phosphorylated RNAP II CTD; however, in S . cerevisiae , triphosphatase and in S . pombe , triphosphatase and guanylyltransferase bind the phosphorylated RNAP II CTD independently 40 , 55 . These enzymes preferred to bind a CTD substrate that was phosphorylated at Ser5 or doubly phosphorylated at Ser2 and Ser5 in the highly conserved heptapeptide repeats Y 1 S 2 P 3 T 4 S 5 P 6 S 7 . T . gondii Rbp1-CTD contains a mixture of 10 heptapeptide YSPxSPx sequences instead of conserved YSPTSPS; however, Ser2 and Ser5 residues are conserved in those 10 heptapeptide repeats. The role of Ser5- 56 and Ser2- 57 phosphorylation has been shown in transcription initiation and elongation, respectively, in T . gondii . Hence, it will be interesting to test which T . gondii capping enzyme interacts with Ser5 and/or Ser2-phosphorylated CTD of Rpb1 58 . Besides, T . gondii triphosphatase and methyltransferase are exceptionally large proteins with N-terminal extensions, suggesting additional functions or more regulatory roles in coordinating cap formation with RNA synthesis 59 , 60 . Also, it is intriguing to test the distribution of TgRNAP II on chromatin without capping to test whether RNAP II transits to the elongation phase, pauses, or transcribes at a low speed or pausing followed by premature transcription termination 61 . Cap sequencing upon TgRT depletion revealed global gene expression defects, resulting in the complete arrest of parasite replication. The most significantly downregulated transcripts were related to chromatin maintenance, structural integrity, and key kinases, which explains why a halt of parasite relocation occurs even with a transient depletion of TgRT. However, the transcripts related to transporter activities, transferase activity, glucose, and glutamate metabolism were upregulated upon loss of capping. The global transcriptome data (ToxoDB) in T . gondii suggest that in TgRT-depleted parasites, most of these downregulated genes were highly expressed, and the genes showed upregulation expressed at low levels. These findings are consistent with the recent study in S . cerevisiae , where the impact of defective capping (depletion of guanylyltransferase) on genome-wide RNA abundance revealed the reduction of highly expressed mRNAs and the accumulation of lowly expressed and more stable mRNAs 62 . In yeast, such cap-defective transcripts are detected by an Npl3-mediated surveillance mechanism that triggers decapping and subsequent RNA degradation. In Toxoplasma , the RNA surveillance mechanism and RNA degradation pathways are yet to be studied. Generally, glucose and glutamate are the sole physiological nutrient sources in carbon metabolism obligatory for parasite growth and survival 63 , 64 . Hence, unsurprisingly, we observed high transcript levels of carbon metabolism genes in cap-defective parasites. Such transcripts are stabilized probably through a posttranscriptional mechanism that maintains the steady-state mRNA levels of these transcripts for such obligatory parasite function. In such a case, the transcript stabilization could outweigh the lowered level of de novo transcription. Stabilizing such transcripts would eventually limit the decline in the transcription rate and ensure some level of advantage to the parasite to survive under deprivation of either carbon source. The reasons for this stability might be attributed to RNA binding proteins, specificity in the decay pathways, other mRNA modifications, or RNA stability elements such that even in the absence of productive transcription 65 , these mechanisms could contribute to maintaining existing mRNA pools of certain genes. This interplay between stabilization and degradation processes is essential for the dynamic regulation of gene expression and might explain the adaptation and survival mechanisms in the parasite. Hence, in the choice to live or die, the parasite prefers to use its energy on basic metabolic processes for its survival than on the process required for its replication, and this explains why DNA replication and packaging genes were downregulated in TgRT-depleted parasites. The complete differences between the tertiary structures, active sites, and chemical mechanisms of the RNA triphosphatase component of the mRNA capping system in pathogenic fungi, viruses, and protozoa and those of their metazoan hosts highlight TPase as a target for anti-infective drug discovery 23 – 25 , 66 . The triphosphate tunnel metalloenzyme (TTM)-type TPases are essential for the growth of S . cerevisiae (ScCet1) 23 , S . pombe (SpPct1) 26 , the human-pathogenic fungus Aspergillus fumigatuts (AfTriA) 27 , and the human-pathogenic protozoa T . brucei (TbCet1) 16 . To our knowledge, it has not been determined whether TTM-type TPase is essential for pathogen growth in the host. Here, we employed an auxin-inducible degron approach to show that Toxoplasma TgRT (TTM-type) is essential for the growth of parasites in both the culture and the mouse host. TgRT showed robust manganese-dependent NTPase activity in an exceptionally wide range of temperatures and pH. The crystal structures of ScCet1 23 and TbCet1 37 revealed that the TTM active site comprises essential amino acids that either coordinate a metal ion or the γ-phosphate or stabilize the tunnel architecture. Most of these active site residues in the triphosphate tunnel are conserved in TgRT, and the alanine mutations of a few of these residues showed a dramatic reduction in the activity, suggesting a similar function as observed for ScCet1 67 and TbCet1 37 in interacting with the 5′-end of the triphosphate RNA substrate. The recent studies on biochemical screening for small-molecule inhibitors showed that TbCet1 could be inhibited using a nanomolar concentration of various classes of phenolic compounds 17 , whereas kribellosides, metabolites from actinomycetes, selectively inhibit ScCet1 68 . These inhibitors displayed great potency and selectivity for the target enzyme, indicating separate screening against pathogen-specific TTM-type TPases. Hence, the high-resolution crystal structure of TgRT, along with screening for small-molecule inhibitors, will provide critical insights into the basis for TgRT inhibition. The identified compounds can then be tested for parasite growth inhibition in the culture and mouse (natural Toxoplasma host). It is tempting to speculate that these parasite-specific inhibitors may not inhibit human (host) thiamine triphosphatase (hTTP), the only known TTM with similar metal binding sites 69 , owing to the presence of alpha helix at the c-terminal end of hTTP, which positions the thiamine in a way that prevents the entry of non-specific substrates like pppRNA, thereby ensuring specificity 70 (Fig. 10 ). Overall, our work presents a detailed characterization of three-component RNA-capping machinery in Toxoplasma , where RNA triphosphatase is an essential component with complete divergence in the structure and catalytic mechanism from human ortholog. Lack of capping mediated through RNA triphosphatase depletion results in perturbation of gene expression, which is detrimental to the parasite, both in vitro and in vivo , and renders RNA triphosphatase an attractive therapeutic target for Toxoplasma infection. Declarations Ethics statement The institutional ethics committee of National Institute of Animal Biotechnology (IAEC/NIAB/2019/48/ASD) has approved using laboratory animals and research protocols. Consent to participate Not applicable Competing interests The authors declare no competing financial or personal interests. Authors' contributions Kalyani Aswale: Data curation, investigation, analysis and writing manuscript. Abhijit S. Deshmukh: Conceptualization, funding acquisition, investigation and writing manuscript. Funding This work was supported by an NIAB core grant (C022) for ASD. Data and materials availability The Cap sequencing data for the TgRTmAID-3HA transgenic parasite line upon 8h of treatment with vehicle or IAA has been deposited in NCBI’s SRA data with accession numbers SRR27549198 (control) and SRR27560546 (Treatment). The illustration in this study was created using BioRender (https://biorender.com/). 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Structure-function analysis of the active site tunnel of yeast RNA triphosphatase. J Biol Chem 276, 17261-17266 (2001). Igarashi M, et al. Kribellosides, novel RNA 5'-triphosphatase inhibitors from the rare actinomycete Kribbella sp. MI481-42F6. J Antibiot (Tokyo) 70, 582-589 (2017). Bettendorff L, Wins P. Thiamine triphosphatase and the CYTH superfamily of proteins. FEBS J 280, 6443-6455 (2013). Delvaux D, et al. Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase. Biochim Biophys Acta 1830, 4513-4523 (2013). Additional Declarations There is NO Competing Interest. Supplementary Files TheCapsequencing.docx Reviewer token Cap-Seq raw data NCOMMS2403581.pdf Reporting summary SupplementaryInformation.docx Supplementary information SupplementaryTable1.docx Supplementary Table 1. Details of primers and yeast strains used in this study. SupplementaryTable2.xlsx Supplementary Table 2. Details of downregulated and upregulated genes determined by cap sequencing. Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → 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-3875304","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268602067,"identity":"273bb299-300e-4071-9858-4ca11f2a7608","order_by":0,"name":"Abhijit Deshmukh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACCR6GAxIGDHIMzAxsEBFm4rQYGJOmBQgMEhsYYFoIAf7ZvQcPWBT8Sd/OzvzsAUPFPbsGdt4D+C25cy4B5LDcnc1s5gYMZ4qTG5j5EvBbcyPHAKxlw2EGMwnGtoRkBmYeA7w65KFa0g0Os38jTosBVEuCwWEesC12BLUY3jkD0mJsuOEwT7lBwpmEBDZCWuRu9xh/lvgjJ29w/vi2Bx8qEuz5+c/g1wICzBIwVgIDQ2IbQfVAwPgBiWNPjI5RMApGwSgYWQAADCA+TMtwixMAAAAASUVORK5CYII=","orcid":"","institution":"National Institute of Animal Biotechnology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Abhijit","middleName":"","lastName":"Deshmukh","suffix":""},{"id":268602068,"identity":"5075df5f-d74a-465c-97ad-bf3279f42525","order_by":1,"name":"Kalyani Aswale","email":"","orcid":"","institution":"National Institute of Animal Biotechnology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kalyani","middleName":"","lastName":"Aswale","suffix":""}],"badges":[],"createdAt":"2024-01-18 09:06:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3875304/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3875304/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-59867-z","type":"published","date":"2025-07-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50056295,"identity":"2390b948-e4a0-4715-ad61-c93cccd2e617","added_by":"auto","created_at":"2024-01-23 17:53:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2009965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of capping enzymes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eToxoplasma\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Immunofluorescence analysis of m\u003csup\u003e7\u003c/sup\u003eG mark in tachyzoites and bradyzoites.\u003cstrong\u003e B.\u003c/strong\u003e Immuno-dot blot analysis of m\u003csup\u003e7\u003c/sup\u003eG levels in the indicated amount of RNA and Toxoplasma genomic DNA (gDNA). EtBr staining serves as a loading control. Tz: tachyzoite and Bz: bradyzoite. Scale bar 5µm.\u003cstrong\u003e C.\u003c/strong\u003e Predicted three capping genes of Toxoplasma (TgRT, TgGT, and TgGMT) with ToxoDB number and CRISPR score. \u003cstrong\u003eD. \u003c/strong\u003eSchematic depicting TgRT: the triphosphatase tunnel (364-728 aa) with conserved amino acids, TgGT: the catalytic domain (111-255 aa) with conserved amino acids and C-terminal domain 330-480 aa), and TgGMT: the conserved SAM binding motif. \u003cstrong\u003eE-H.\u003c/strong\u003e \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e capping enzymes functionally complement yeast (\u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e) counterparts. \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae \u003c/em\u003eΔ\u003cem\u003ecet1\u003c/em\u003e, or Δ\u003cem\u003eceg1\u003c/em\u003e, or Δ\u003cem\u003eabd1\u003c/em\u003e strain was transformed with pYES3 (TRP1) plasmid containing TgRT, TgGT or TgGMT gene. Triple deletion yeast strain was transformed with TgRT, TgGT, and TgGMT genes containing plasmids. A control transformation was performed with the pYES3 plasmid lacking an insert. Single Trp\u003csup\u003e+\u003c/sup\u003e transformants were patched to agar plates lacking tryptophan (-Trp) and then patched on agar medium containing FOA (-Trp+FOA). FOA-resistant colonies were picked and streaked on -Trp+FOA agar medium.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/ea8ffc109fb82d2bfcc9d5df.jpg"},{"id":50056716,"identity":"192c46e8-e2a4-4fab-9deb-0da6b6663ce8","added_by":"auto","created_at":"2024-01-23 18:02:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1272738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTriphosphatase activity of TgRT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C.\u003c/strong\u003e The expression and localization of TgRT. Coomassie blue-stained SDS-PAGE gel of recombinant full-length His-TgRT protein (A), Western blot analysis of TgRT expression levels in tachyzoites and bradyzoites (B). Immunofluorescence analysis in RH/ME49 parasites with α-TgRT antibody (C). Loading controls: TgAldolase (TgALD) - parasite proteins, TgSAG1 and TgBAG1, tachyzoite- and bradyzoite stage-specific markers, respectively. Tz: tachyzoite and Bz: bradyzoite. Scale bar 5µm.\u003cstrong\u003e D.\u003c/strong\u003e Manganese-and Magnesium-dependent activity of TgRT. TPase reaction mixtures contained 1.5 mM [γ-\u003csup\u003e32\u003c/sup\u003eP]ATP, 0.5 μg of TgRT, and either MgCl\u003csub\u003e2\u003c/sub\u003e or MnCl\u003csub\u003e2\u003c/sub\u003e as specified or no added divalent cation. The reaction products were analyzed by TLC and visualized by autoradiography. The positions of [γ-\u003csup\u003e32\u003c/sup\u003eP]ATP and \u003csup\u003e32\u003c/sup\u003ePi are indicated. \u003cstrong\u003eE.\u003c/strong\u003e TgRT titration. TPase reaction mixtures contained 5 mM MnCl\u003csub\u003e2\u003c/sub\u003e and TgRT as specified. \u003cstrong\u003eF.\u003c/strong\u003e TgRT enzyme activity with different divalent cations. TPase reactions were set up as described in (A). The reaction mixtures contained 1.5 mM of [γ-\u003csup\u003e32\u003c/sup\u003eP]ATP, 0.5 μg of TgRT, and 5 mM divalent cation as specified. The Pi release was calculated as a function of a divalent cation. \u003cstrong\u003eG.\u003c/strong\u003e TgRT enzyme activity at various incubation durations. TPase reactions were incubated for indicated time points, and Pi release was quantified. \u003cstrong\u003eH.\u003c/strong\u003e TgRT activity at different temperatures. TPase reactions were incubated at specified temperatures, and Pi release was estimated. \u003cstrong\u003eI.\u003c/strong\u003e TgRT activity at variable pH. TPase reactions were performed in the Tris buffer with pH as indicated, and Pi release was estimated. \u003cstrong\u003eJ.\u003c/strong\u003e TPase activity of TgRT with different NTP substrates. The reactions were set up as described in (A). The reaction mixtures contained 0.5 μg of TgRT, 2 mM MnCl\u003csub\u003e2\u003c/sub\u003e, and either [γ -\u003csup\u003e32\u003c/sup\u003eP]ATP or [α-\u003csup\u003e32\u003c/sup\u003eP]ATP or [α-\u003csup\u003e32\u003c/sup\u003eP]UTP as specified. \u003cstrong\u003eK.\u003c/strong\u003e Coomassie blue-stained SDS-PAGE gel of recombinant His-tag WT and mutant TgRT proteins (E366A and E726A) as indicated. \u003cstrong\u003eL.\u003c/strong\u003e TPase activity of WT and mutant TgRT. The reaction mixtures contained 1.5 mM of [γ-\u003csup\u003e32\u003c/sup\u003eP]ATP, 2 mm MnCl2, and either WT or mutant proteins as indicated. The Pi release was plotted as a function of the input enzyme. Graphical representation for Pi release is Mean ± S.D. (\u003cem\u003en\u003c/em\u003e=3 replicates). Adjusted p values: *\u0026lt;0.05; *** \u0026lt;0.001; ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/791bde76e2dfedb8b514b244.jpg"},{"id":50056294,"identity":"32011d9e-faa0-4960-8b54-3dbac269ac68","added_by":"auto","created_at":"2024-01-23 17:53:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1052506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGuanylyltransferase activity of TgGT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C\u003cstrong\u003e.\u003c/strong\u003e The expression and localization of TgGT. Coomassie blue-stained SDS-PAGE gel of recombinant full-length His-TgGT protein (A), Western blot analysis of TgGT expression levels in tachyzoites and bradyzoites (B). Immunofluorescence analysis in RH/ME49 parasites with α-TgGT antibody (C). Loading controls: TgAldolase (TgALD) - parasite proteins, TgSAG1 and TgBAG1, tachyzoite- and bradyzoite stage-specific markers, respectively. Tz: tachyzoite and Bz: bradyzoite. Scale bar 5µm.\u003cstrong\u003e D. \u003c/strong\u003eManganese-and Magnesium-dependent GTase activity of TgGT. The reaction mixtures contained 1 mM [α-\u003csup\u003e32\u003c/sup\u003eP]GTP, 0.5 μg of TgGT, and either MgCl\u003csub\u003e2\u003c/sub\u003e or MnCl\u003csub\u003e2\u003c/sub\u003e as specified or no added divalent cation. The reactions were resolved on SDS PAGE gel, visualized by autoradiography and the TgGT-GMP adduct was assessed. \u003cstrong\u003eE.\u003c/strong\u003e TgGT titration. The reaction mixtures contained 2 mM MnCl\u003csub\u003e2\u003c/sub\u003e and TgGT as specified. \u003cstrong\u003eF.\u003c/strong\u003e Coomassie blue-stained SDS-PAGE gel of recombinant His-tag WT and mutant TgGT proteins (K133A, T134A, D135A, and G136A) as indicated. \u003cstrong\u003eG.\u003c/strong\u003e GTase activity of WT and mutant TgGT. The reaction mixtures contained 1 mM of [α-\u003csup\u003e32\u003c/sup\u003eP]GTP, 2 mm MnCl\u003csub\u003e2\u003c/sub\u003e, and either WT or mutant proteins as indicated. TgGT-GMP intermediate was visualized by autoradiography (left panel). The TgGT-GMP intermediate quantification shown in the graph represents the average of three experiments with S.D. (right panel).\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/ba2529b3f6b087e305e293d1.jpg"},{"id":50056296,"identity":"e24d0532-2693-46c6-85f0-7c7b95cc69e2","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1067923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGuanine-N7 methyltransferase activity of TgGMT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C.\u003c/strong\u003e The expression and localization of TgGMT. Coomassie blue-stained SDS-PAGE gel of recombinant His\u003csub\u003e6-\u003c/sub\u003eTgGMT\u003csub\u003e716-1283\u003c/sub\u003e protein (A), Western blot analysis of TgGMT expression levels in tachyzoites and bradyzoites (B). Immunofluorescence analysis in RH/ME49 parasites with α-TgGMT antibody (C). Loading controls: TgAldolase (TgALD) - parasite proteins, TgSAG1 and TgBAG1, tachyzoite- and bradyzoite stage-specific markers, respectively. Tz: tachyzoite and Bz: bradyzoite. Scale bar 5µm.\u003cstrong\u003e D.\u003c/strong\u003e Experimental workflow showing steps involved to generate 32 mer (N\u003csub\u003e32\u003c/sub\u003e) RNA variants, such as triphosphate RNA (pppN\u003csub\u003e32\u003c/sub\u003eRNA), diphosphate RNA (ppN\u003csub\u003e32\u003c/sub\u003eRNA), Guanylated RNA (GpppN\u003csub\u003e32\u003c/sub\u003eRNA), 7-methylguanosine RNA (m\u003csup\u003e7\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003eRNA). \u003cstrong\u003eE.\u003c/strong\u003e Immuno-dot blot analysis of RNA with α-m\u003csup\u003e7\u003c/sup\u003eG antibody. RNA variants were generated by incubating IVT RNA with or without capping enzymes as specified. Vaccinia capping enzyme (VCE) was used as a positive control. Methylene blue stain serves as an RNA loading control. Graphical representation is Mean ± S.D. (\u003cem\u003en\u003c/em\u003e=3 replicates) (bottom panel). \u003cstrong\u003eF.\u003c/strong\u003e SAM dependent RNA methylation. A capping reaction was performed with or without Sinefungin at the specified concentration.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/4d1cccbeb19a4c5bdabdaa87.jpg"},{"id":50056298,"identity":"f7fd2fde-f06b-45ca-a5a2-a28fa807dd6c","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1349329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCap-dependent translation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egondii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C.\u003c/strong\u003e The expression and localization of TgeIF4E. Coomassie blue-stained SDS-PAGE gel of recombinant full-length TgeIF4E-His protein (A), Western blot analysis of TgeIF4E expression levels in tachyzoites and bradyzoites (B). Immunofluorescence analysis in RH/ME49 parasites with α-TgeIF4E antibody (C) Tz: tachyzoite and Bz: bradyzoite. Scale bar 5µm. \u003cstrong\u003eD-F.\u003c/strong\u003e Microscale thermophoresis analysis of labeled TgeIF4E with pppN\u003csub\u003e32\u003c/sub\u003eRNA (D), GpppN\u003csub\u003e32\u003c/sub\u003eRNA (E), and m\u003csup\u003e7\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003eRNA (F). \u003cstrong\u003eG.\u003c/strong\u003e Experimental workflow showing generation luciferase (FLuc) RNA variants with or without 5’-cap and 3’-poly(A) tail, such as pppFLuc, pppFLuc-[A]\u003csub\u003en\u003c/sub\u003e, and m\u003csup\u003e7\u003c/sup\u003eGpppFLuc-[A]\u003csub\u003en\u003c/sub\u003e. VCE generated m\u003csup\u003e7\u003c/sup\u003eGpppFluc-[A]\u003csub\u003en\u003c/sub\u003e was used as a positive control. \u003cstrong\u003eH.\u003c/strong\u003e \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e tachyzoites were transfected with luciferase RNAs synthesized with and without 5’-cap and 3’-poly(A) tails as specified. Parasites were transfected with m\u003csup\u003e7\u003c/sup\u003eGpppFluc-[A]\u003csub\u003en\u003c/sub\u003e generated by VCE as a positive control or mock transfected with no RNA as a negative control. Luciferase activity was determined 20 h post-transfection. The readings were normalised with VCE RNA (m\u003csup\u003e7\u003c/sup\u003eGpppFLuc-[A]\u003csub\u003en\u003c/sub\u003e). Representation is Mean ± SD (\u003cem\u003en\u003c/em\u003e=3 replicates). One way ANOVA was used to calculate the p value. Adjusted p values: *\u0026lt;0.05; *** \u0026lt;0.001; ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/616ffb52776cdad9935edf93.jpg"},{"id":50056307,"identity":"e56e6ed7-db63-4de2-b9dc-343ac30d23d8","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1991881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of TgRT depletion on RNA capping and parasite growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Strategy for tagging of TgRT protein in RH-TIR1-3FLAG parental line. A mAID-3HA tag and HXGPRT selection cassette were integrated into the C-terminus of the TgRT protein by CRISPR/Cas9-mediated homologous recombination. \u003cstrong\u003eB.\u003c/strong\u003e Diagnostic PCR from genomic DNA of RH-TIR1 and TgRT-mAID-3HA parasites to check the 3′ integration of mAID-3HA in the gene locus. \u003cstrong\u003eC.\u003c/strong\u003e Western blot analysis of TgRT-mAID-HA expression levels in tachyzoites with or without IAA as indicated time-points using anti-HA antibodies. \u003cstrong\u003eD.\u003c/strong\u003e Immunofluorescence analysis of TgRT-mAID-HA grown with IAA or vehicle for 1 h. Scale bar 5µm. \u003cstrong\u003eE and F.\u003c/strong\u003e Immuno-dot blot analysis of m\u003csup\u003e7\u003c/sup\u003eG levels in the indicated amount of RNA extracted from TgRT-mAID-HA parasite cultured in the presence of IAA or vehicle for 8 h. \u003cstrong\u003eG.\u003c/strong\u003e TgRT-mAID-3HA parasites were transfected with TgTub-FLuc plasmid DNA. Luciferase activity was measured at indicated time points post-transfection. \u003cstrong\u003eH.\u003c/strong\u003e Parasite replication. TgRT-mAID-3HA parasites grown in HFF monolayers for 18 h with IAA or vehicle. Number of parasites per vacuole was counted for 50 random vacuoles and plotted as a percentage of a total number of vacuoles. \u003cstrong\u003eI.\u003c/strong\u003e TgRT-mAID-3HA parasites were grown with IAA or vehicle for 8 h. Number of parasites with defective morphology per vacuole were counted and plotted as a percentage of normal and abnormal parasites. \u003cstrong\u003eJ.\u003c/strong\u003e Crystal violet stained images of plaques formed by RH-TIR1 and TgRT-mAID-3HA parasites on HFF monolayer treated with IAA or vehicle. \u003cstrong\u003eK and L.\u003c/strong\u003e Quantification of plaque numbers (K) and plaque areas (L) from three independent experiments (Mean ± SD). \u003cstrong\u003eM and N.\u003c/strong\u003e Plaque assay. TgRT-mAID-3HA parasites were grown on HFF monolayers with IAA or vehicle at the indicated time points, media were replaced with normal medium, and parasite growth was determined by plaque assays. \u003cstrong\u003eO.\u003c/strong\u003e Parasite invasion. The graph shows the percent invasion of RH-TIR1 and RT- mAID-3HA parasites on HFF monolayer. \u003cstrong\u003eP.\u003c/strong\u003e Parasite egress. The graph shows the percent egress of RH-TIR1 and RT- mAID-3HA parasites cultured with IAA or vehicle followed by calcium ionophore treatment. IF analysis was performed to check intact or collapsed vacuoles. The graph representation is Mean ± SD (\u003cem\u003en\u003c/em\u003e=3 replicates). Student’s t-test was used to calculate the p-value: *\u0026lt;0.05; *** \u0026lt;0.001; ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/a8ebf6a26f1c0f79f68bbd7e.jpg"},{"id":50056304,"identity":"0fbaa78f-e14e-4a96-856c-8354f6cc4b9f","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1530898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of TgRT depletion on RNA capping in Toxoplasma.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Experimental workflow showing steps involved to enrich capped RNA. Total RNA was isolated from TgRT-mAID-3HA parasites grown with IAA or vehicle. RNA was treated with the indicated enzymes and subsequently used for library construction and RNA sequencing. \u003cstrong\u003eB.\u003c/strong\u003e Agarose gel image showing rRNA levels in -/+ enzyme treatment in TgRT-mAID-3HA parasites grown with IAA or vehicle. \u003cstrong\u003eC.\u003c/strong\u003e Immuno-dot blot showing m\u003csup\u003e7\u003c/sup\u003eG RNA levels in -/+ enzyme treatment in TgRT-mAID-3HA parasites grown with IAA or vehicle. Methylene blue stain serves as a loading control. \u003cstrong\u003eD.\u003c/strong\u003e \u003cem\u003ek\u003c/em\u003e-means clustering of 2000 transcripts. A total four clusters (A, B, C, and D) were defined. The colour key ranges from -2 to +2 (green to red). \u003cstrong\u003eE.\u003c/strong\u003e Pie chart showing overall percentage distribution of upregulated, downregulated, and unchanged capped transcripts. \u003cstrong\u003eF.\u003c/strong\u003e Volcano plot showing differentially expressed genes in TgRT-mAID-3HA parasites with IAA or vehicle. \u003cstrong\u003eG,H.\u003c/strong\u003e Gene ontology analysis of downregulated (G), and upregulated (H) expressed genes with respect to cellular components and biological processes.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/eef42aa9f651d652b07df388.jpg"},{"id":50056719,"identity":"2a4f18c3-cc86-4bd8-8264-0cb41db6e864","added_by":"auto","created_at":"2024-01-23 18:02:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1116861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepletion of TgRT \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protects mice from lethal toxoplasmosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B.\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e assessment of TgRT-mAID-3HA knockdown. BALB/c mice were challenged with 50 TgRT-mAID-3HA parasites intraperitoneally and treated with IAA or vehicle from day 2 to 6. On day 5 and 6, mice were sacrificed, and peritoneal exudate cells were collected for IF microscopy. Fixed cells were probed for parasites (rabbit α-TgIMC1 and α -rabbit IgG Alexa Fluor 594) and RT-mAID-3HA (mouse α-TgHA and α -mouse IgG Alexa Fluor 488). Scale bar, 5 μm. \u003cstrong\u003eC\u003c/strong\u003e. Experimental design of \u003cem\u003ein vivo \u003c/em\u003etest of TgRT essentiality. Related to Figures 8A and 8B (\u003cem\u003en\u003c/em\u003e=10 mice per group and \u003cem\u003en\u003c/em\u003e = 2 trials). \u003cstrong\u003eD.\u003c/strong\u003e Survival curve of BALB/c mice infected with 50 TgRT-mAID-3HA parasites intraperitoneally and treated with IAA or vehicle for 15 days. The Gehan-Breslow-Wilcoxon test was used to compare differences between the survival curves, p \u0026lt; 0.0001 (IAA vs control). \u003cstrong\u003eE. \u003c/strong\u003eMean body weight ± SD of BALB/c mice infected with 50 TgRT-mAID-3HA parasites intraperitoneally and treated with IAA or vehicle for 15 days.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/62c06a384e2b95ebb69d0c00.jpg"},{"id":50056301,"identity":"67516f3a-18bd-4e60-8414-e4c60842892d","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1613164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural conservation of RNA triphosphatase.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Predicted structure of TgRT (361-729aa) using Collabfold. \u003cstrong\u003eB.\u003c/strong\u003e The structure contains a ten-stranded anti-parallel β-barrel with three α-helices surrounding the active-site tunnel.\u003cstrong\u003e C.\u003c/strong\u003e The predicted structure of TgRT in green was superimposed on \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae \u003c/em\u003eCet1 colored in yellow (1D8H).\u003cstrong\u003e D. \u003c/strong\u003eConservation of active site residues of ScCet1, TgRT, TcCet1, and hTTP. \u003cstrong\u003eE.\u003c/strong\u003e The predicted structure of TgRT in green was superimposed on \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ecruzi\u003c/em\u003e Cet1 colored in blue (6L7W). \u003cstrong\u003eF.\u003c/strong\u003eThe predicted structure of TgRT in green was superimposed on human thiamine triphosphatase colored in red (3TVL).\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/51b62b30a996066c3ed91f96.jpg"},{"id":50056720,"identity":"b1d1f19f-eb36-4ddd-bea6-7609b691bb95","added_by":"auto","created_at":"2024-01-23 18:02:00","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1007513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe illustration shows the effect of lack of capping mediated by TgRT depletion on the parasite. \u003c/strong\u003eThe intricate mRNA capping process orchestrates a precise equilibrium in mRNA levels, thereby ensuring the regular growth and replication of the parasite, evident in both \u003cem\u003ein vitro\u003c/em\u003e (A) and \u003cem\u003ein vivo\u003c/em\u003e (B) settings. The absence of TgRT, an essential mRNA capping protein, results in impaired mRNA capping and leads to parasite replication arrest (C) and rendering the parasites non-viable, ultimately preventing lethal toxoplasmosis (D) Leveraging insights from both in vitro and in vivo investigations, the strategic use of a small molecule inhibitor, specifically designed to target the TgRT RNA entry tunnel (E), emerges as a promising approach to block TgRT effectively. This inhibitor approach could mimic the observed effects in (D) and offer a potential avenue for controlling lethal \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e infection.\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/9cf0500318c14c32ade92c01.jpg"},{"id":85832401,"identity":"a32fb79a-1192-410d-80de-3afaa31416cf","added_by":"auto","created_at":"2025-07-02 07:55:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15604377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/2b1e3aff-b77e-4822-a8a1-22a2e166f562.pdf"},{"id":50056293,"identity":"0ac73ae2-b294-44c7-9ae5-d46d2486e616","added_by":"auto","created_at":"2024-01-23 17:53:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13720,"visible":true,"origin":"","legend":"\u003cp\u003eReviewer token Cap-Seq raw data\u003c/p\u003e","description":"","filename":"TheCapsequencing.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/b412954f1f7cd90726dc435a.docx"},{"id":50056299,"identity":"49e58643-72d9-48d8-ad57-2d1ea9133d9d","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1666250,"visible":true,"origin":"","legend":"\u003cp\u003eReporting summary\u003c/p\u003e","description":"","filename":"NCOMMS2403581.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/c0b8d3954f18d2b52497f7aa.pdf"},{"id":50056306,"identity":"412a612b-b4d2-4d36-bc4e-0d617591d3fc","added_by":"auto","created_at":"2024-01-23 17:54:00","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2353415,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary information\u003c/p\u003e","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/df67b461217f14f4a1e81d54.docx"},{"id":50056718,"identity":"53f1af63-8ac4-4ae4-846a-a1f67c982b0e","added_by":"auto","created_at":"2024-01-23 18:02:00","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16510,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. Details of primers and yeast strains used in this study.\u003c/p\u003e","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/2e91cb472268e1877dad1aa7.docx"},{"id":50056717,"identity":"937937c7-d069-4190-bbfd-7474f4cce0c3","added_by":"auto","created_at":"2024-01-23 18:02:00","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":18677,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2. Details of downregulated and upregulated genes determined by cap sequencing.\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3875304/v1/a810275204aeaa50a3b529b8.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eToxoplasma gondii\u003c/em\u003e is an extremely successful obligate intracellular apicomplexan parasite that causes lifelong chronic infections in almost all warm-blooded animals, including humans, and severe disease in fetuses and immunocompromised individuals\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. An estimated one-third of the global human population is chronically infected with \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Following strong host immune response during the acute infection, the rapidly multiplying tachyzoites differentiate into a slow-growing encysted bradyzoite and develop chronic infection\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A fast conversion of bradyzoites into tachyzoites leads to severe mortality in immunosuppressed individuals if not treated. The standard treatment drugs for toxoplasmosis are few and have severe side effects on long-term use, target the tachyzoite stage of the parasite, and are ineffective against encysted bradyzoite in the tissues, necessitating the identification of new drug targets and therapeutics\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe complex multi-host life cycle of \u003cem\u003eToxoplasma\u003c/em\u003e involves changes between life stages with distinct morphologies, metabolisms, and reproductive niches. Parasite utilizes an intricate transcriptional, post-transcriptional, and epigenetic network to regulate gene expression required to thrive in these rapidly changing environments and hosts\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The growing evidence suggests that RNA processing, especially co-transcriptional and post-transcriptional mechanisms, is crucial for regulating gene expression during life cycle stage transitions and environmental adoption; however, the underlying mechanism is yet to be discovered in Apicomplexa parasites.\u003c/p\u003e \u003cp\u003eIn eukaryotes, 5\u0026rsquo; mRNA capping is the first and one of the essential co-transcriptional modifications to produce mature mRNA\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The 5' cap is essential throughout the life cycle of the mRNA in coordinating various functional processes accomplished by interacting with the cap-biding complex (CBC). The nuclear CBC facilitates splicing, polyadenylation, and export into the cytoplasm, whereas the cytoplasmic CBC containing eukaryotic initiation factor 4F (eIF4F) binds to the cap and recruits the 40S ribosomal subunit to initiate translation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAll eukaryotic mRNA contains a 5' cap structure of 7-methylguanosine (m\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG) linked \u003cem\u003evia\u003c/em\u003e 5\u0026prime; to 5\u0026prime; triphosphate bridge to the first transcribed nucleotide (m\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN). The m7GpppN cap structure is formed by a series of three enzymatic steps: (i) hydrolysis of the 5\u0026prime; triphosphate (pppN) end of the nascent RNA to a diphosphate (ppN) by RNA triphosphatase ; (ii) transfer of a guanine monophosphate (GMP) nucleotide to the 5\u0026prime;-diphosphate RNA (GpppN-guanosine cap) by RNA guanylyltransferase; and (iii) addition of a methyl group to the N7 amine of the guanine cap (m7GpppN) by guanine-N7 methyltransferase. These three catalytic activities, collectively called \u0026lsquo;capping enzymes,' are encoded by separate genes in yeast, whereas in metazoans, the first two capping steps are catalyzed by a single enzyme consisting of two functional domains, the N-terminal triphosphatase and the C-terminal guanylyltransferase\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The structures and mechanisms of the mammalian and fungal capping enzymes have been well elucidated; however, studies on the capping enzymes of protozoa\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, except \u003cem\u003eTrypanosoma\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, are mostly limited to their biochemical characterization.\u003c/p\u003e \u003cp\u003eWhile guanylyltransferase (GTase) and methyltransferase (guanine-N7 MTase) are conserved, RNA triphosphatase (TPase) has a distinct structure and mechanism of action among eukaryotes. Consequently, RNA triphosphatases (TPase) are classified into two families: i) the divalent cation-dependent TPase of triphosphate tunnel metalloenzyme (TTM) family found in fungi\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and protozoa\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and ii) the divalent cation-independent TPase of metazoans and plants of cysteine-phosphatase superfamily found in metazoan and plants\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The TTM-type TPases are essential for growth in fungi\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eTrypanosoma brucei\u003c/em\u003e protozoan parasite\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and as a result, RNA triphosphatases are considered potential drug targets for these infections. The TPase protein essentiality studies are carried out only in tissue culture; however, its essentiality has never been tested for pathogen survival in the host. Additionally, the impact of a lack of capping in the TPase-deficient pathogens on genome-wide M7G-capped RNA abundance, mRNA expression, turnover, and stability has never been studied.\u003c/p\u003e \u003cp\u003eHere, we report a detailed characterization of Toxoplasma capping enzymes and investigate the consequences of RNA triphosphatase depletion on transcript homeostasis and parasite survival. Toxoplasma has a functional three-component capping system consisting of separate TTM-type RNA triphosphatase (TgRT), guanylyltransferase, and guanine-N7 methyltransferase. The m\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG RNA generated using capping enzymes bind to the translation initiator factor, TgeIF4E, and is successfully translated into protein in the parasite. We used an auxin-inducible degron method to generate TgRT conditional knockdown parasites and show that rapid depletion of TgRT leads to downregulation of global m7G-capped transcripts of essential genes, which resulted in the death of the parasite in the culture and in the mouse host. Lastly, we predicted the structure of TgRT, which will enable us to develop an effective inhibitor with minimal or no impact on structurally similar proteins in the host.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eParasite culture\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eT. gondii\u003c/em\u003e tachyzoites of RH and ME49 strains were maintained in human foreskin fibroblast cells (HFFs, ATCC) in DMEM containing 10% foetal bovine serum, 10 \u0026micro;g/ml gentamicin, 1% penicillin-streptomycin, and 2 mM L-glutamine at 37\u0026deg;C and 5% CO2. Tachyzoite to bradyzoite stage differentiation was carried out by incubating the ME49 tachyzoites in bradyzoite induction medium (RPMI pH 8.2) at 37\u0026deg;C for 5 days without CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eCloning, expression and purification of TgRT, TgGT, TgGMT, and TgeIF4E\u003c/h2\u003e\n\u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e codon-optimised ORF of \u003cem\u003eT. gondii\u003c/em\u003e RT, GT, GMT\u003csub\u003e2146\u0026thinsp;\u0026minus;\u0026thinsp;3849\u003c/sub\u003e (subscript denotes nucleotide coordinates), and eIF4E were synthesised by Life Technologies. All three genes were initially cloned into the pMK-RQ vector (Life Technologies) between \u003cem\u003eNde\u003c/em\u003eI-\u003cem\u003eEcoR\u003c/em\u003eI sites, which were further subcloned into the pET-28a or pET-21a (Novagen, USA) (Supplementary Table\u0026nbsp;1), and recombinant proteins were expressed in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e BL21 Rosetta as N-terminal (TgRT, TgGT, and TgGMT) and C-terminal (TgeIF4E) 6-xHis-tag and purified on a nickel-nitrilotriacetic acid-agarose resin column as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Briefly, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e transformed with pET28a-TgRT/TgGT/TgGMT/TgeIF4E was grown in 10 ml of Luria-Bertani (LB) medium supplemented with 50 \u0026micro;g/ml kanamycin and 34 \u0026micro;g/ml chloramphenicol overnight at 37\u003csup\u003eo\u003c/sup\u003eC. Subsequently, 10 ml of the overnight culture was added to 1000 ml of LB containing the same antibiotics and incubated at 37\u003csup\u003eo\u003c/sup\u003eC with vigorous shaking. When the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6, IPTG was added to the culture to a final concentration of 1 mM, and the cells were further incubated at 25\u003csup\u003eo\u003c/sup\u003eC for 16 h. The cells were then harvested by centrifugation, and the pellets were resuspended in 50 ml of lysis buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 300 mM NaCl, 100 \u0026micro;g/ml lysozyme, 10 mM imidazole, 0.1% Triton X-100, and 0.3 mM PMSF, pH 8.0). After centrifugation, the protein was purified from the supernatant with the use of a Ni\u003csup\u003e2+\u003c/sup\u003e-NTA agarose (Qiagen) and step-eluted with 1-ml aliquots of 20\u0026ndash;300 mm imidazole in lysis buffer. The quality of the eluted protein was analysed using SDS-PAGE. The appropriate elutes containing the protein were then dialyzed in 1X PBS and stored at -80\u0026deg;C. TgRT mutants (E366A and E726A) and TgGT mutants (K133A, T134A, D135A, and G136A) were generated using a specific set of primers (Supplementary Table\u0026nbsp;1) following the Stratagene (210515) site-directed mutagenesis protocol. Recombinant mutant protein purification was performed as for wild-type proteins.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003ePolyclonal antibody raising\u003c/h2\u003e\n\u003cp\u003eMouse polyclonal antibodies to recombinant TgRT, TgGT, TgGMT, and TgeIF4E were generated by primary injection with 30 \u0026micro;g of purified recombinant protein in Freund's complete adjuvant (#F5881, Sigma) followed by four boosts of 20 \u0026micro;g each in Freund's incomplete adjuvant (#F5506, Sigma) at 2-week intervals. Serum was collected after day 60 post immunization. Polyclonal antibodies for Tg- IMC1\u003csup\u003e29\u003c/sup\u003e, SAG1\u003csup\u003e30\u003c/sup\u003e, BAG1\u003csup\u003e31\u003c/sup\u003e, CST1\u003csup\u003e30\u003c/sup\u003e, and Aldolase (ALD) antibodies were used from previous studies.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunoblotting\u003c/h2\u003e\n\u003cp\u003eFilter-purified 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e parasites were suspended in SDS-PAGE sample buffer and boiled for 10 min before being run on a single lane of a 10% polyacrylamide gel. The gel was then transferred to a 0.2 \u0026micro;m PVDF membrane (BioRad) using a Trans-Blot System (BioRad) for 12 h at 30 V. The PVDF membrane was blocked in 5% (w/v) non-fat milk in PBS for 60 min before being probed with a primary antibody (\u0026alpha;-TgRT/TgGT/TgGMT/TgeIF4E-1:500; \u0026alpha;HA-1:5,000; \u0026alpha;-TgSAG1/TgBAG1/TgALD-1:2,000) in non-fat milk overnight at 4\u0026deg;C. The blot was washed 3x with PBS plus Tween-20 detergent (PBST; 0.1% Tween-20) before probing with either HRP-conjugated \u0026alpha;-rabbit or \u0026alpha;-mouse-IgG (Invitrogen). The blot was washed and developed using the Clarity Western ECL kit (BioRad) and visualised on a ChemiDoc Imager (Biorad).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunofluorescence (IF) staining\u003c/h2\u003e\n\u003cp\u003eHFFs were grown on glass coverslips until confluent and subsequently infected with \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e RH/ME49/TgRT-mAID-HA tachyzoites. The infected HFFs were fixed with methanol-free 4% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS for 15 min at room temperature, and blocked with 5% (w/v) bovine serum albumin (BSA, Sigma) in PBS for 60 min at RT. Primary and secondary antibodies were diluted in 1% (w/v) BSA in PBS. Samples were first incubated with the primary antibody (\u0026alpha;-TgRT/TgGT/TgGMT/TgeIF4E-1:100; \u0026alpha;HA-1:1,000; \u0026alpha;TgIMC1/TgSAG1/TgCST1 -1:2,000) at 25\u003csup\u003eo\u003c/sup\u003eC for 60 min, washed 5x with PBS, and then incubated with fluorescent secondary antibodies (1:1000) and 4',6-diamidino-2-phenylindole (DAPI; 300 nM) at 25\u003csup\u003eo\u003c/sup\u003eC for 60 min. Secondary antibodies (Invitrogen) were conjugated to either Alexa Fluor (AF) green or red fluorophores and specific to the species of primary antibody used. Samples were then washed 5x with PBS before mounting the coverslip on a glass slide using Vectashield medium (Vector Laboratories). IF staining was visualised using a Leica confocal microscope with a 100X oil immersion objective. Images were processed using las x software (Leica Microsystems). A similar protocol was employed for bradyzoite IF staining.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eImmuno dot-blot\u003c/h2\u003e\n\u003cp\u003eRNA and genomic DNA were isolated from filter-purified parasites using RNeasy kit (Qiagen) and DNeasy Blood and Tissue kit (Qiagen), respectively. Indicated amounts of nucleic acids were spotted onto the Hybond-N\u0026thinsp;+\u0026thinsp;membrane (Sigma) and fixed to the membrane with a UV crosslinker (Bioanalytik Jena). The dried membrane was stained with ethidium bromide or methylene blue stain for 15 min at room temperature to stain the spotted nucleic acids. The membrane was blocked for 30 min in 5% (w/v) skim milk in PBS and washed 3x with PBS. The membrane was first incubated with \u0026alpha;-m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG antibody (Sigma, 1:1000) at 4\u003csup\u003eo\u003c/sup\u003eC for 12 hrs, washed 5x with PBS, and then incubated with HRP-conjugated anti-mouse secondary antibody (Invitrogen) at 25\u003csup\u003eo\u003c/sup\u003eC for 60 min. The membrane was washed and developed using the Clarity Western ECL kit and visualised on a ChemiDoc Imager. The Cap sequencing experiment followed a similar procedure to determine the m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG level of RNA samples treated with RppH and XrnI from control and IAA-treated parasites.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA triphosphatase (RT) activity\u003c/h2\u003e\n\u003cp\u003eRT activity was assayed\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e by quantifying the release of \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003ePi from \u0026gamma;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP-labelled ATP. Standard reaction mixtures (10 \u0026micro;l) containing 50 mM Tris HCl (pH 7.5), 5 mM DTT, 5 mM MnCl\u003csub\u003e2\u003c/sub\u003e/MgCl\u003csub\u003e2\u003c/sub\u003e/ZnCl\u003csub\u003e2\u003c/sub\u003e/CaCl\u003csub\u003e2\u003c/sub\u003e, 1.5 mM ATP, and TgRT protein as specified were incubated for 30 min at 30\u0026deg;C. The reaction mixtures were applied to a polyethyleneimine-cellulose thin-layer chromatography (TLC) plate, which was developed with 0.75 M potassium phosphate (pH 4.3). The release of \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003ePi from [\u0026gamma;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP was quantitated by scanning the TLC plate with a Phosphor Imager (Typhoon, Cytiva). The 5 mM MnCl\u003csub\u003e2\u003c/sub\u003e and 500 ng TgRT protein were used to test the time-dependent, temperature-dependent, and pH-dependent RNA triphosphatase activity. RNA triphosphatase activity in the presence of different NTPs was assayed using 1.5 mM [\u0026gamma;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP or 1.5 mM [\u0026alpha;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP or 1.5 mM [\u0026alpha;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]UTP, and 500 ng TgRT protein.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eGuanylyltransferase (GT) assay\u003c/h2\u003e\n\u003cp\u003eGT activity was measured\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e in reaction mixtures (20 \u0026micro;l) containing 50 mM Tris HCl (pH 8.0), 5 mM DTT, 5 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 1 mM [\u0026alpha;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]GTP, and TgGT protein (0.5\u0026ndash;4 \u0026micro;g) that were incubated for 30 min at 37\u0026deg;C. The reactions were stopped by adding SDS-PAGE sample buffer and boiling for 10 min. The samples were electrophoresed through a 10% polyacrylamide gel, and the GT-GMP intermediate complex was determined by autoradiography using a phosphor imager.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003ePreparation of m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003e RNA\u003c/h2\u003e\n\u003cp\u003eTemplate dsDNA (N\u003csub\u003e32\u003c/sub\u003e mer) and RNA variants were prepared as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The dsDNA (Supplementary Table\u0026nbsp;1) was used to generate pppN\u003csub\u003e32\u003c/sub\u003e RNA using MEGAScript T3 Transcription Kit (Ambion). The pppN\u003csub\u003e32\u003c/sub\u003e RNA was treated with TgRT (as described in the TgRT activity) to obtain ppN\u003csub\u003e32\u003c/sub\u003e RNA, and the ppN\u003csub\u003e32\u003c/sub\u003e RNA was treated with TgGT (as described in the TgGT activity) to obtain GpppN\u003csub\u003e32\u003c/sub\u003e RNA. To obtain m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN32 RNA from GpppN\u003csub\u003e32\u003c/sub\u003e RNA, GpppN\u003csub\u003e32\u003c/sub\u003e RNA was incubated with TgGMT protein in a reaction mixture containing capping buffer for 30\u0026deg;C for 3 h. The vaccinia capping enzymes (VCE, NEB) was used in the reaction to convert pppN\u003csub\u003e32\u003c/sub\u003e RNA to m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003e RNA. The obtained RNA samples were heated at 95\u003csup\u003eo\u003c/sup\u003eC for 2 min and purified using an RNA cleanup and concentrator kit (Zymo).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eLuciferase expression assay\u003c/h2\u003e\n\u003cp\u003eThe firefly luciferase gene was amplified (from the pmirGLO plasmid, Promega) by PCR using the forward primer (Supplementary Table\u0026nbsp;1) containing the T3 promoter sequence and the starting sequence from the luciferase gene. The obtained DNA was gel-purified and subjected to IVT using the MEGAScript T3 Transcription Kit (Ambion). The reaction was incubated at 37\u0026deg;C for 3 h, treated with DNaseI at 37\u0026deg;C for 1 h, and IVT luciferase RNA was purified using the RNA cleanup kit. The luciferase RNA (ppp-FLuc RNA) was treated sequentially with TgRT, TgGT, and TgGMT proteins or VCE to obtain m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-FLuc RNA. The Poly(A) tailing of ppp-Fluc RNA-[A]\u003csub\u003en\u003c/sub\u003e or m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-FLuc RNA-[A]\u003csub\u003en\u003c/sub\u003e was performed using \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Poly(A) Polymerase (NEB# M0276). Around 20 \u0026micro;g of each RNA variant was used to transfect 10\u003csup\u003e7\u003c/sup\u003e RH parasites using the Gene Pulser Xcell Total System (Biorad, #1652660). Transfected parasites were immediately transferred to a new flask containing a confluent HFF monolayer. Parasites were harvested 18 hrs post-infection, and luciferase activity was determined using the Luciferase Reporter Assay System (Promega, # E1910).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eMicroscale thermophoresis (MST) assay\u003c/h2\u003e\n\u003cp\u003eMST assays were performed with a Monolith NT.115 (Nanotemper). His-tagged TgRT protein was labelled with RED-MALEIMIDE 2nd generation dye according to the protocol provided by Nanotemper. Dilutions of the RNA variants (pppN32, GpppN32, and m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN32) in a range of 0.18 nM to 600 nM were prepared in in a buffer containing 50 mM HEPES, pH 7.2, 1 mM EDTA, 1 mM DTT, and 100 mM KCl. The reactions were incubated at RT for 15 min, followed by the loading of reaction mixtures into glass capillaries (Nanotemper). The fluorescence intensity for each reaction was measured by keeping 40% infrared laser power and 60% light-emitting diode. The fluorescence values were analysed using the Affinity Analysis software version 2.3 (Nano Temper) to determine the binding affinity (dissociation constant: KD) between TgeIF4E and the RNA variants used.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eGeneration of auxin-inducible TgRT-mAID-HA transgenic parasites\u003c/h2\u003e\n\u003cp\u003eTgRT-mAID-HA transgenic parasites were generated by CRISPR/Cas9-mediated site-specific gene editing using the \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e line RH TIR1-3FLAG\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. A CRIPSR/Cas9 plasmid with a specific guide RNA (gRNA) targeting the 3\u0026rsquo; end of RT was generated from the pSAG1::Cas9-U6::sgUPRT plasmid (Addgene, #54467) by a Q5 Hot Start site-directed mutagenesis kit (NEB) and primers (Supplementary Table\u0026nbsp;1) to induce double-strand DNA breaks and direct the insertion of the PCR fragment. The PCR fragment containing the mAID-3HA tag and the HXGPRT selection was amplified from the vector pTUB1:YFP-mAID-3HA using the primers (Supplementary Table\u0026nbsp;1) with 40 bp of homology with the 3\u0026prime; end of RT and Q5 polymerase (NEB) to facilitate direct insertion of the PCR fragment and double homologous recombination. Ten \u0026micro;g each of TgRT-mAID-HA amplicon and pSAG1::Cas9-U6::sgRT were transfected into 10\u003csup\u003e7\u003c/sup\u003e \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e RH TIR1-3FLAG parasites by electroporation using the Gene Pulser Xcell Total System (Biorad, #1652660). Transfected parasites were drug-selected using mycophenolic acid (25 \u0026micro;g/ml) and xanthene (50 \u0026micro;g/ml) for two growth cycles before cloning out by serial dilution. Endogenous tagging of TgRT-mAID-HA was verified using sequencing, diagnostic PCR, immunoblotting, and IF staining. The auxin-inducible degradation of TgRT-mAID-HA was tested by culturing these parasites in a medium containing 500 \u0026micro;M indole-3-acetic acid (IAA) (Sigma, I2886), followed by immunoblotting and IF staining using \u0026alpha;-HA antibody.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003ePlaque assay\u003c/h2\u003e\n\u003cp\u003eHFFs were grown in 6-well plates until confluent and subsequently infected with 100 parasites per well. After 24 h, the medium was removed and the wells were treated with 500 \u0026micro;M IAA or an equivalent volume of MeOH vehicle. Plates remained undisturbed for 5 days before the infected HFFs were fixed with 100% ice-cold MeOH for 20 min and stained with a 1% crystal violet solution for 20 min. Plaques (zone of lysis: white) could be visualised against intact cells (purple). The plaque areas were quantified using ImageJ. Three independent experiments were performed with similar results. For the rescue experiment, a similar plaque assay procedure was followed; however, the IAA-containing medium was replaced with normal parasite medium at the indicated time points.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eReplication\u003c/h2\u003e\n\u003cp\u003eHFFs were grown on glass coverslips in a 6-well plate until confluent and subsequently infected with 10\u003csup\u003e4\u003c/sup\u003e parasites per well. After 12 h, the medium was removed, and the wells were treated with 500 \u0026micro;M IAA or an equivalent volume of MeOH vehicle. 18 hours later, infected HFFs were fixed, permeabilized, stained with \u0026alpha;-TgIMC1 (1:2,000) and DAPI, and subjected to IF staining. The number of parasites per vacuole was determined by counting the parasites from 50 random vacuoles. A total of three independent replicates were performed. A similar procedure was followed to count the morphologically defective parasites.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eInvasion\u003c/h2\u003e\n\u003cp\u003eParasites grown in HFF monolayers were treated with vehicle or IAA for 14 h to deplete TgRT-mAID-3HA protein. Parasites were then harvested, and added to HFF monolayers cultured on coverslips (10\u003csup\u003e5\u003c/sup\u003e parasites/well) for 20 min at 37C in the presence of the vehicle or IAA. Infected HFFs were fixed and stained with rabbit \u0026alpha;-TgIMC1 (1:2,000). Following 3x washes with PBS, infected HFFs were permeabilized and stained with mouse \u0026alpha;-TgIMC1 (1:2,000) and DAPI. After 3x washes with PBS, infected HFFs were stained with AF-conjugated secondary antibodies (anti-mouse IgG AF 488 and anti-rabbit IgG AF 568) and DAPI. Parasites (green/red) were counted from thirty random fields. The relative efficiency of attachment and invasion of IAA-treated parasites was expressed as a mean percentage of the control treatment from three independent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eEgress\u003c/h2\u003e\n\u003cp\u003eHFFs were grown on glass coverslips in a 6-well plate and infected with 10\u003csup\u003e4\u003c/sup\u003e parasites per well. After 8 h, the medium was replaced with DMEM containing 500 \u0026micro;M IAA or MeOH vehicle. 24 hours later, parasite egress was triggered by the addition of 3 \u0026micro;M calcium ionophore A23187 to infected HFFs for 2 min. The infected HFFs were fixed, permeabilized, and stained with \u0026alpha;-TgGRA2 (1:1000) and \u0026alpha;-TgIMC1 (1:2000). A total of 150 vacuoles from each experiment (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) were examined to count the intact or collapsed vacuoles. Vacuoles containing\u0026thinsp;\u0026gt;\u0026thinsp;2 parasites were considered intact. Anti-GRA2 (NR-50260) monoclonal antibody was procured from BEI resources.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eCAP sequencing\u003c/h2\u003e\n\u003cp\u003eRT-mAID-HA parasites (5 x 10\u003csup\u003e7\u003c/sup\u003e) were harvested from infected HFFs grown in DMEM containing 500 \u0026micro;M IAA or MeOH vehicle for 8 h. Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) and subjected to enzymatic treatments, followed by RNA purification using the RNA cleanup kit. First, 5\u0026micro;g RNA was treated with RppH (NEB) in NEB buffer 2 at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 h, followed by treatment with Xrn1 (NEB) in NEBuffer 3 at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 h. The treatment of these two enzymes ensured enrichment of 5\u0026rsquo;m7G and 5\u0026rsquo;OH RNAs and marked depletion of rRNA. Subsequently, RNA was treated with RppH (NEB) in Thermopol buffer at 37 \u003csup\u003eo\u003c/sup\u003eC for 1 h to convert m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-RNA to p-RNA, which was confirmed by dot blot using the \u0026alpha;-m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG antibody. The quality of recovered RNA was assessed using tapestation 4150. The obtained RIN values were \u0026lt;\u0026thinsp;2, confirming successful rRNA depletion in the RNA samples. The RNA samples were quantified using a Qubit flurometer (Thermofisher #Q33238). The cDNA libraries were prepared using the Truseq Kit (Illumina), followed by the sequencing of paired end reads on the Illumina NovaSeq 6000 platform. The read quality was examined using MultiQC and FastaQC. The reads were mapped onto the Toxoplasma reference genome (GCF_000006565.2) using STAR v2.7.9a, followed by transcript quantification by RSEM. Differential expression analysis (DEA) was performed using DESeq2. The k-means cluster analysis was performed using iDEP.96. The outputs from feature counts (DESeq2 and k-means clustering) were represented using RStudio. Gene ontology analysis was performed using ToxoDB.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eMouse infection\u003c/h2\u003e\n\u003cp\u003eThe auxin-inducible degradation of protein in mice was performed as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e with some modifications. To test TgRT depletion in vivo, 6-week BALB/c male mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4) were injected intraperitoneally (i.p.) with 50 tachyzoites of RH TgRT-mAID-HA (2 groups). From the second day of post-infection (pi), of two groups, one group was given IAA, and the other group was administered an equivalent volume of MeOH vehicle in the drinking water containing 5% sucrose. The IAA was administered in two ways: i) in drinking water (0.5 mg/ml) and ii) by oral gavage (12.5 mg/mL). The total treatment period was 4 days (2 days pi to 5 days pi). On day 5 and 6 pi, 2 mice each were sacrificed by CO\u003csub\u003e2\u003c/sub\u003e asphyxiation, and the peritoneal exudate cells (PECs) were collected and examined by IF staining using \u0026alpha;-HA and \u0026alpha;-TgIMC1 antibodies to test the TgRT protein depletion. After successful depletion of TgRT-mAID-HA in IAA-administered mice, a long-term (30-day) survival experiment was performed by following a similar experimental procedure. In the 30-day survival experiment, each group had 10 mice (2 groups RH TgRT-mAID-HA); mice were weighed and monitored daily; PECs were collected on days 6 and 12; IAA treatment was given for 15 days in the surviving mice; and survived mice were sacrificed on day 30. Relative weight loss was calculated based on the initial body weight on the day of infection. The overall experimental setup and procedures used are indicated within the relevant figures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eStructure predictions\u003c/h2\u003e\n\u003cp\u003eThe alphafold structure\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e of TgRT (S8F0G9) was obtained from ToxoDB. The disordered region was manually removed and amino acid sequence corresponding to 361\u0026ndash;729 aa, which consists of 10\u0026beta; barrel tunnels was used as input sequence for structure prediction using GoogleCollab notebook\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFol\u003c/span\u003e\u003c/span\u003ed.ipynb#scrollTo\u0026thinsp;=\u0026thinsp;rowN0bVYLe9n. The obtained structure of RT was superimposed with PDB structures of RNA triphosphatase of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (1D8H)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e (6L7W)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and thiamine triphosphatase\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e of \u003cem\u003eHomo sapiens\u003c/em\u003e (3VTL) using PyMol (v2.x).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eYeast complementation\u003c/h2\u003e\n\u003cp\u003eFor complementation assays, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae \u0026Delta;cet1\u003c/em\u003e, \u003cem\u003e\u0026Delta;ceg1\u003c/em\u003e, \u003cem\u003e\u0026Delta;abd1\u003c/em\u003e, and three genes chromosomal copy deletion mutant strains (Supplementary Table\u0026nbsp;1) carrying the wild-type copy in a plasmid with \u003cem\u003eURA\u003c/em\u003e marker were utilized\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Full-length \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii rt\u003c/em\u003e, and \u003cem\u003egt\u003c/em\u003e and C-terminus of \u003cem\u003egmt\u003c/em\u003e genes were cloned (Supplementary Table\u0026nbsp;1) into pYES3/CT vector between \u003cem\u003eKpn\u003c/em\u003eI-\u003cem\u003eEcoR\u003c/em\u003eI, \u003cem\u003eBamH\u003c/em\u003eI-\u003cem\u003eXho\u003c/em\u003eI, and \u003cem\u003eKpn\u003c/em\u003eI-\u003cem\u003eEcoR\u003c/em\u003eI, respectively. The \u003cem\u003ecet\u003c/em\u003e, \u003cem\u003eceg\u003c/em\u003e, and \u003cem\u003eabd\u003c/em\u003e, yeast mutant stains were transformed with respective plasmid carrying respective \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e gene or empty plasmid. Triple deletant mutant strain was transformed with plasmids carrying all three \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e genes or empty vector. Transformants were selected on SD \u0026ndash;Trp plates with or without 5-Fluoroorotic Acid (5-FOA).\u003c/p\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eData analyses\u003c/h2\u003e\n\u003cp\u003eAll data analyses, including graph preparation and statistics, were performed using GraphPad Prism 9. Immunoblot quantification was performed using ImageJ (Version 6).\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e. \u003cstrong\u003egondii\u003c/strong\u003e \u003cstrong\u003eencodes three separate mRNA capping enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the presence of 7-methylguanosine (m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG) cap in \u003cem\u003eToxoplasma\u003c/em\u003e, we performed immunofluorescence analysis using an anti-m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG antibody in the asexual stages (tachyzoite and bradyzoite) of the parasite. Analysis of intracellular parasites revealed predominant punctate staining for m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG at the nuclear periphery and cytoplasm of tachyzoite (Tz) and bradyzoite (Bz) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). To detect whether the m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG mark is present on RNA, we performed an immuno-dot blot using an anti-m7G antibody on total RNA and genomic DNA (gDNA) extracted from filter-purified tachyzoites. The results demonstrated that the m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG mark was exclusively present in RNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Collectively, these results show that m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG cap RNA is a feature of \u003cem\u003eToxoplasma\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo identify capping enzymes, which could add m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG structure to RNA, we performed BLASTP homology searches of the \u003cem\u003eToxoplasma\u003c/em\u003e genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://toxodb.org/toxo/\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"Underline\"\u003e)\u003c/span\u003e using amino acid sequences of yeast Cet1, Ceg1, and Abd1 proteins as queries. The search analysis revealed three separate RNA capping enzymes in \u003cem\u003eToxoplasma\u003c/em\u003e that are similar to yeast. The identified candidate \u003cem\u003eToxoplasma\u003c/em\u003e RNA triphosphatase, guanylyltransferase, and guanine-N7 methyltransferase were named TgRT (TGME49_224650), TgGT (TGME49_305320), and TgGMT (TGME49_272720), respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). The MEGA analysis was performed to determine how closely the \u003cem\u003eToxoplasma\u003c/em\u003e capping enzymes (TgCEs) are related to the alveolates' and other eukaryotes' capping proteins. Phylogenetic analysis revealed a distinction between two apicomplexan classes, \u003cem\u003eConoidasida\u003c/em\u003e and \u003cem\u003eAconoidasida\u003c/em\u003e, for all three capping proteins. Members of the \u003cem\u003eSarcocystidae\u003c/em\u003e family were found to encode the largest triphosphatase and methyltransferase proteins (Supplementary Fig.\u0026nbsp;1A-C).\u003c/p\u003e\n\u003cp\u003eThe putative TgRT gene encodes a 920-aa polypeptide and has characteristic features of fungal triphosphatases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, including two glutamate-containing metal-binding motifs, homologs of \u0026beta; strands that comprise the active site tunnel, and conserved hydrophilic amino acids required for catalysis. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, Supplementary Fig.\u0026nbsp;2). The putative TgGT is a 509-aa protein contains two conserved domains, a nucleotidyl transferase (NTase) domain and a C-terminal oligonucleotide-binding domain (OB)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The NTase domain has six conserved motifs (I, III, IIIa, IV, V, and VI)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;3) with a lysine-containing KxDG motif I (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), which comprises the active site of GTP-binding and nucleotidyl transfer\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The putative TgGMT encodes 1283-aa polypeptide with large N-terminal extension and conserved glycine-rich sequence in the SAM-binding motif (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Among all three capping enzymes, TgRT and TgGMT are significantly larger than yeast's TPase and guanine-N7 MTase; however, each of the three \u003cem\u003eToxoplasma\u003c/em\u003e capping enzyme orthologue is essential for parasites according to a genome-wide CRISPR screen (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting that M\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG capping of RNA is important and required for parasite fitness.\u003c/p\u003e\n\u003cp\u003eOwing to the sequence similarity between \u003cem\u003eToxoplasma\u003c/em\u003e capping enzymes (TgCEs) and yeast enzymes, we performed yeast complementation to test whether TgCEs function in the cap-synthetic pathway and sustain the growth of yeast cells that lack one or more capping enzymes. We separately cloned the \u003cem\u003eTgRT\u003c/em\u003e, \u003cem\u003eTgGT\u003c/em\u003e, and \u003cem\u003eTgGMT\u003c/em\u003e genes into a yeast 2\u0026micro; \u003cem\u003eTRP1\u003c/em\u003e pYES3 plasmid, and the function of each of these genes was tested by plasmid shuffle in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae \u0026Delta;cet1 or \u0026Delta;ceg1 or \u0026Delta;abd1\u003c/em\u003e cells that contain respective gene on a \u003cem\u003eCEN URA3\u003c/em\u003e plasmid. The mutant strain cannot survive on a medium containing 5-FOA, a drug that selects against the \u003cem\u003eURA3\u003c/em\u003e plasmid unless it is transformed with a second plasmid containing a functional homolog from another source. We found that 2\u0026micro; \u003cem\u003eTgRT\u003c/em\u003e, \u003cem\u003eTgGT\u003c/em\u003e, and \u003cem\u003eTgGMT\u003c/em\u003e supported the growth of \u003cem\u003e\u0026Delta;cet1\u003c/em\u003e, \u0026Delta;ceg1, and \u003cem\u003e\u0026Delta;abd1\u003c/em\u003e cells, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). Similarly, 2\u0026micro; \u003cem\u003eTgRT\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eTgGT\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eTgGMT\u003c/em\u003e supported the growth of triple mutant yeast cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). These results demonstrate that \u003cem\u003eToxoplasma\u003c/em\u003e encodes biologically active capping enzymes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eToxoplasma\u003c/strong\u003e \u003cstrong\u003eRNA Triphosphatase TgRT shows metal-dependent triphosphatase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain insight into the expression, localization, and biochemical function of TgRT, full-length His\u003csub\u003e6\u003c/sub\u003e-TgRT protein of ~\u0026thinsp;100 kDa was purified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) and used to generate specific anti- TgRT antibodies. The expression and localization studies revealed that TgRT is robustly expressed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) in both the asexual stages and localized in the nucleus of the tachyzoite and bradyzoite stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), as demonstrated using anti-TgRT antibodies. Further, we tested the triphosphatase activity of TgRT using a detailed biochemical characterization. The recombinant wild type (WT) TgRT catalyzed the release of \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003ePi from [\u0026gamma;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP in the presence of manganese (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD); however, the ATP hydrolysis was ineffective in the presence of magnesium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). The extent of ATP hydrolysis was proportional to TgRT concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). No ATP hydrolysis was observed without divalent cation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). ATP activity was nominal in the presence of calcium or zinc (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). The optimal ATPase activity of TgRT was detected as early as 5 min and remained stable for 30 min (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). The enzyme activity of TgRT was similar from 20\u003csup\u003eo\u003c/sup\u003eC to 70\u003csup\u003eo\u003c/sup\u003eC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). TgRT was found to be catalytically active in a wide pH range (5.5\u0026ndash;10) with an optimal activity from pH 6.5 to 8.0 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI). We also tested the specificity for NTP hydrolysis using two different triphosphorylated nucleoside substrates. The rate of release of \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003ePi from [\u0026gamma;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP was similar to the rate of conversion of [\u0026alpha;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ATP to [\u0026alpha;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]ADP and [\u0026alpha;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]UTP to [\u0026alpha;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]UDP in a parallel reaction mixture containing the same concentration of TgRT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Two glutamate residues corresponding to the metal binding sites of the tunnel were replaced by alanine (E366A and E726A) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eK), and enzyme activities were compared. ATPase activity of the E366A mutant was 50% of the activity of wild-type TgRT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eL), whereas in comparison, E726A showed\u0026thinsp;\u0026lt;\u0026thinsp;10% activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eL). Less activity in the mutant proteins was due to a change in the secondary structure, as demonstrated by circular dichroism (Supplementary Fig.\u0026nbsp;3). Together, these results demonstrated that TgRT belongs to the family of triphosphate tunnel metalloenzymes (TTMs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of\u003c/strong\u003e \u003cstrong\u003eToxoplasma\u003c/strong\u003e \u003cstrong\u003eGuanylyltransferase TgGT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length His\u003csub\u003e6\u003c/sub\u003e-TgGT protein of ~\u0026thinsp;60 kDa was purified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA) and used to generate anti-TgGT antibodies. TgGT is robustly expressed in the asexual stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB) and localized in the nucleus of the tachyzoite and bradyzoite stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). All known guanylyltransferases accomplish nucleotidyl transfer through a covalent enzyme-(lysyl-N)-GMP intermediate that can be detected by label transfer from [\u0026alpha;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]GTP to the enzyme. To determine the guanylyltransferase activity of TgGT, protein was incubated with [\u0026alpha;-\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003eP]GTP and a divalent cation, which resulted in the formation of an SDS-stable\u0026thinsp;~\u0026thinsp;60 kDa enzyme-GMP intermediate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). TgGT activity requires a divalent cation cofactor, either manganese or magnesium; however, enzyme activity was more effective in the presence of manganese than magnesium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Enzyme-guanylate formation was linear with respect to TgGT concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). We mutated conserved Lys (essential for GMP interaction during the guanylyltransferase reaction for GTases)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, Tyr, Glu, and Gly residues to alanine (K133A, T134A, D135A, and G136A) of TgGT and compared enzyme activities (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). None of the mutant proteins showed GTase activity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG); however, after long autoradiographic exposure, a trace of residual activity was observed for T134A (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). No activity in the TgGT mutant proteins was due to a change in the secondary structure, as demonstrated by circular dichroism (Supplementary Fig.\u0026nbsp;4). We conclude that the observed guanylyltransferase activity is intrinsic of TgGT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of\u003c/strong\u003e \u003cstrong\u003eToxoplasma\u003c/strong\u003e \u003cstrong\u003eguanine-N7 methyltransferase TgGMT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHis\u003csub\u003e6\u0026minus;\u003c/sub\u003eTgGMT\u003csub\u003e716\u0026thinsp;\u0026minus;\u0026thinsp;1283\u003c/sub\u003e (subscript denotes amino acid coordinates) protein of ~\u0026thinsp;60 kDa was purified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) and used to generate anti-TgGMT antibodies. The expression and localization studies showed TgGMT expressed in the asexual stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) and primarily localized in the nucleus of the tachyzoite and bradyzoite stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). To test the guanine-N7 methyltransferase activity of TgGMT, we first generated 32 mer RNA (pppN\u003csub\u003e32\u003c/sub\u003eRNA) using \u003cem\u003ein vitro\u003c/em\u003e transcription and sequentially treated with TgRT to generate ppN\u003csub\u003e32\u003c/sub\u003e, TgGT to generate GpppN\u003csub\u003e32\u003c/sub\u003e, and TgGMT in the presence of methyl donor S-adenosyl methionine (SAM) to generate m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003e, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD. The individual reaction was spotted on the membrane and capping of RNA substrate was determined using anti-m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG antibody. The immune-blot analysis revealed that TgGMT could successfully add m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG to the guanylated RNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Vaccinia Capping Enzyme (VCE) was used as a positive control to generate m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). The specificity of TgGMT to use SAM was tested using sinefungin, a structural analog of SAM and inhibitor of methyltransferases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Immunoblot analysis using anti-m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG antibody revealed that sinefungin inhibits m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003e synthesis in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). Together, these results demonstrate that TgCEs are biochemically active and function in a cap-synthetic pathway in \u003cem\u003eToxoplasma\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCap-dependent translation\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe eukaryotic translation initiation factor, eIF4E, binds to the m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG cap of mRNA and initiates translation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Similarly, we wanted to test whether \u003cem\u003ein vitro\u003c/em\u003e-generated capped RNA could be recognized by eIF4E-like protein and productively translated into protein in \u003cem\u003eToxoplasma\u003c/em\u003e. BLASTP search using \u003cem\u003ePlasmodium\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and human\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e eIF4E revealed three eIF4E-like homologues (TGME49_223410, TGME49_315150, and TGME49_312560) in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e genome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Of these three homologue, TGME49_223410 showed highest amino acid similarity, including conserved residues required for m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG interaction with \u003cem\u003ePlasmodium\u003c/em\u003e (e score: 1e-69) and human protein (e score: 1e-14) (Supplementary Fig.\u0026nbsp;6). While the eIF4E protein is highly conserved in eukaryotes, TgeIF4E clusters with fungi and other protozoan parasites, and the metazoans form a separate clan (Supplementary Fig.\u0026nbsp;7). Hence, we name this putative protein TgeIF4E, a 225-aa protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). The full-length TgeIF4E-His\u003csub\u003e6\u003c/sub\u003e protein of ~\u0026thinsp;26 kDa was purified (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) and used to generate specific antibodies. TgeIF4E is robustly expressed in the asexual stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) and, as expected, localized in the cytoplasm of the tachyzoite and bradyzoite stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eTo determine the interaction between TgeIF4E with capped or non-capped RNA variants generated using TgCEs, a microscale thermophoresis (MST) assay was performed using fluorescently labeled TgeIF4E. As measured, TgeIF4E showed strong binding affinity (K\u003csub\u003eD\u003c/sub\u003e=8.01\u0026thinsp;\u003cspan class=\"Underline\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1 nM) towards m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eGpppN\u003csub\u003e32\u003c/sub\u003eRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF) and no binding affinity was measured for pppN\u003csub\u003e32\u003c/sub\u003eRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) and GpppN\u003csub\u003e32\u003c/sub\u003eRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results confirm the m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG cap specificity of TgeIF4E. Further, to evaluate m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG RNA were utilized by \u003cem\u003eToxoplasma\u003c/em\u003e to promote translation, we generated luciferase transcript variants \u003cem\u003ein vitro\u003c/em\u003e with and without 5\u0026rsquo;-m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG cap (using TgCEs) and 3\u0026rsquo;-poly(A)tails (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). We found that luciferase activity was detected after transfection of capped and polyadenylated RNA (m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-FLuc-[A]\u003csub\u003en\u003c/sub\u003e), but not after transfected with either only IVT RNA (pppFLuc) or capped RNA (m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-FLuc) or polyadenylated RNA (pppFLuc-[A]\u003csub\u003en\u003c/sub\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). As a positive control, 5\u0026rsquo;capped and 3\u0026rsquo;polyadenyaled FLuc RNA was generated using VCE and PolyA polymerase and transfected. However, after comparing the luciferase activity, we found that TgCEs are less effective than VCE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). Together, these results demonstrate that \u003cem\u003ein vitro\u003c/em\u003e-generated capped RNA using TgCEs can be utilized by \u003cem\u003eToxoplasma\u003c/em\u003e to promote translation \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n\u003ch2\u003eDepletion of TgRT impairs overall m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG levels and arrest parasite replication\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eToxoplasma\u003c/em\u003e RNA triphosphatase is a metalloenzyme unique to the parasite. To understand better the role of this protein in the transcription-associated processes, we endogenously tagged TgRT at the C-terminus with mini auxin-inducible degron (mAID) fused with three copies of HA (3HA) epitope (TgRT-mAID-3HA) in RH strain parasites expressing TIR1, which allows rapid degradation of the TgRT-mAID-HA protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA) upon the addition of indole 3-acetic acid (IAA). The resulting TgRT-mAID-HA strain was confirmed by diagnostic PCR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Western blot analysis using anti-HA antibody revealed complete loss of the TgRT-mAID-HA protein in as little as 1 h following addition of 500 \u0026micro;M IAA in the culture medium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Using IF analysis, we also observed no staining for TgRT-mAID-HA in the 1 h IAA treated parasites (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Consistent with the role in the mRNA capping, depletion of TgRT for 8 h diminished m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG capped RNA in the parasite (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Initially, we checked the m7G levels of total parasite RNA of control vs treated parasites at 1 h, 4 h, and 8 h; however, we could observe a significant decline in m7G levels after 8 h of TgRT depletion. Hence, we selected an 8 h IAA treatment period for all the further experiments. The effect of TgRT depletion on productive translation was examined by transfecting TgRT-mAID-HA parasites with Luciferase plasmid. As measured, the luciferase activity was decreased after 4 h, and a drastic reduction in the activity was observed after 8 h of RT depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). Collectively, these results show that depletion of TgRT leads to reduced RNA capping in the parasite.\u003c/p\u003e\n\u003cp\u003eWe next examined the effect of TgRT depletion on parasite-specific processes. The complete arrest of parasite replication was observed in the TgRT-depleted parasites tested using a standard parasite counting assay (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH). A significant number of parasites displayed morphological defects upon TgRT depletion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI). The impact of TgRT depletion on parasite growth was tested using plaque assays. Unlike the parental strain, TgRT-depleted parasites produced no visible plaques (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eJ-L). To determine whether parasites can recover from a transient loss of TgRT expression, we conducted a plaque assay that used six different IAA treatment regimens (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eM). After an initial 24 h of growth, parasites were treated with IAA or vehicle for 1/2/4/8/12/24 h; at this point, the media was replaced with fresh IAA (or vehicle) and incubated for 5 days. The number of plaques formed was similar for 1 h IAA or vehicle-treated parasites (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eM and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eN). Two hours of IAA-treated TgRT-mAID-HA parasites showed a 50% reduction in the number of plaques formed, whereas no plaques were observed when treated for 4 h or 8 h or 12 h, or 24 h IAA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eM and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eN). These results indicate that parasites could not recover after depletion of capping for \u0026gt;\u0026thinsp;4 h. The depletion of TgRT significantly decreased the invasion efficiency of the parasite (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eO); however, no difference was observed in the ability of parasites to egress upon inducing egress using calcium ionophore (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eP). Together, these data provide compelling evidence that TgRT is essential for \u003cem\u003eToxoplasma\u003c/em\u003e viability and proliferation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n\u003ch2\u003eDepletion of TgRT perturbs gene expression\u003c/h2\u003e\n\u003cp\u003eGiven that M\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG RNA capping governs overall RNA metabolism, we reasoned that the loss of parasite viability upon TgRT depletion could be due to a defect in the capped RNA abundance and gene expression. We investigated the consequences of TgRT depletion (the absence of capping) on gene expression using cap sequencing, which was aimed at capturing all the m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG capped RNA population to provide a transcriptome-wide profile that will reveal the identity and relative levels of capped RNA. The m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-capped RNAs were enriched using sequential enzyme treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA) on 8 h IAA- or vehicle-treated TgRT-mAID-HA parasites (in biological duplicates). In the first step for the enrichment capped RNA, total RNAs were treated sequentially with RppH (in the presence of NEBbufer2) and Xrn1 to remove rRNA and 5\u0026rsquo;-monophosphate-ended RNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Subsequently, RNAs were treated with RppH in the presence of Thermopol buffer to convert M\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-capped RNA (decapping) to monophosphate-ended RNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). These decapped RNAs were subjected to library preparation and sequencing, and the obtained transcriptomic data were utilized for differential gene expression analysis (DESeq). The gene expression data from two biological experiments were analyzed using a k-means clustering algorithm. Clustering of 2000 transcripts revealed four clusters (A, B, C, and D) with similar expression profiles in two biological replicates of IAA or vehicle samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). The significantly altered capped transcripts were identified by edgeR (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.005; fold change\u0026thinsp;\u0026ge;\u0026thinsp;1), resulting in 185 and 186 genes whose abundance was decreased and increased, respectively, upon depletion of TgRT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). To understand what processes the differentially expressed genes might be involved in, we performed gene ontology (GO) enrichment analyses (Supplementary Table\u0026nbsp;2). We could identify clear terms for cellular components (CC) and biological processes (BP) for the significantly dysregulated genes. The multiple GO terms were identified for the downregulated genes but were mostly related to \u0026lsquo;DNA packaging\u0026rsquo; and \u0026lsquo;cell membrane\u0026rsquo;. The most significant downregulated genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH) were histones (H1 like protens, H2A1, H2Ba, H2Bb, H2Ax, H3, and H4) and inner membrane complex proteins (IMC16, IMC17, IMC20, IMC22, IMC26, IMC34, IMC35). Transcripts related to protein phosphorylation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG) were found to be significantly downregulated (Rhoptry kinases-ROPs, aurora kinase, calcium-dependent protein kinase). However, transcripts related to transporter activities (phosphate transporters, MC family transporter) and transferase activity (glucosamine transferases, dehydrogenases, esterases) were found to be significantly upregulated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH). Further analysis revealed glucose (PYK1, ENO1, GAPDH1, ENR, and ACC1) and glutamate metabolism (glutathione synthase, glutamate cysteine ligase) pathways upregulated upon loss of capping (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n\u003ch2\u003eDepletion of TgRT protects mice from lethal toxoplasmosis\u003c/h2\u003e\n\u003cp\u003eTgRT protein is essential for parasite fitness in tissue culture; however, to test its essentiality in establishing an infection in the host, we performed mouse infection studies. First, we tested whether TgRT-mAID-HA could be depleted \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). Mice were infected with 50 tachyzoites of RH TgRT-mAID-HA parasites intraperitoneally and then treated orally with 200 mg/kg/day IAA\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e or vehicle from day 2 to 4 post-infection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). On day 5 and 6 pi, the mice were sacrificed and peritoneal exudate cells (PECs) were collected for IF microscopy. Parasites from IAA-treated mice showed depleted TgRT-mAID-HA levels but normal levels of a control protein, TgIMC1 as compared to parasites from the vehicle-treated mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). This experiment confirmed the successful \u003cem\u003ein vivo\u003c/em\u003e depletion of TgRT-mAID-HA protein as early as 3 days after IAA treatment. Next, to determine the effect of prolonged depletion of TgRT on parasite survival in vivo, mice were infected with RH TgRT-mAID-HA parasites and then treated with IAA or vehicle orally for 15 days to deplete TgRT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC). The depletion TgRT was confirmed by IF microscopy in one of the sacrificed mice at 5 day of IAA treatment (data not shown). All mice receiving the vehicle control treatment succumbed to lethal toxoplasmosis by day 11 post-infection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). Conversely, IAA treatment (\u003cem\u003ei.e\u003c/em\u003e., TgRT depletion) rescued all mice from lethal toxoplasmosis, indicating that TgRT is necessary for acute infection in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). Severe morbidity and complete mortality were seen in the control treatment group compared to the IAA treatment group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). By day 2 pi, the control group showed weight loss, a sign of illness, and continued to lose up to a quarter of their initial weight by the time of death. The IAA treatment group showed no weight loss. Suppressing TgRT expression during the acute phase of infection effectively blocked replication of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e since discontinuation of IAA treatment following day 15 did not result in morbidity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE), mortality (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD), or parasite presence (tested by collecting PECs -data not shown) as monitored for an additional 15 days. Overall, these results show the essential role of TgRT for parasite survival and replication in the mouse host.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural analysis of\u003c/strong\u003e \u003cstrong\u003eT\u003c/strong\u003e. \u003cstrong\u003egondii\u003c/strong\u003e \u003cstrong\u003eRNA triphosphatase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tunnel family RNA triphosphatases are potential antiinfective targets owing to the complete divergence in structures and mechanisms of the RNA triphosphatases of the unicellular pathogen and the mammalian host. We showed that the mechanism of action of TgRT is different from its host counterpart; however, we have yet to know the structural details of this protein, which can help develop a parasite-specific inhibitor. To gain insight into the structure of TgRT, we first attempted to perform comparative modeling of TgRT with known PDB structures of other RNA triphosphatases; however, we failed to obtain the structure owing to poor (\u0026lt;\u0026thinsp;40%) sequence homology. Next, we retrieved Alphafold predicted structure of TgRT using the AlphaFold database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/entry/S8F0G9\u003c/span\u003e\u003c/span\u003e). The obtained structure showed a moderate to high confidence score (the per-residue model confidence score: 40\u0026ndash;95) for the region corresponding to the triphosphate tunnel (361-729aa) and a high predicted error (PAE) for the other regions of TgRT (Supplementary Fig.\u0026nbsp;8A and 8B). Finally, using a ColabFold, the structure of the triphosphate tunnel region (361-729aa) was predicted (Supplementary Fig.\u0026nbsp;8C). The predicted structure comprised 10 anti-parallel \u0026beta; barrels (green) with 3 \u0026alpha; helices (blue) surrounding the active tunnel site (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB). Structural comparison with \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e RNA triphosphatase (Cet1) revealed a nearly identical structure (for triphosphatase tunnel) of the two enzymes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC) with 9 of 15 side chain positions important for Cet1 activity conserved in TgRT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD). The triphosphatase tunnel structure of TgRT also showed high structural similarity, including active site residues with the recently deciphered crystal structure of Trypanosoma cruzi RNA triphosphatase. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eE). Next, we checked whether any host protein has similar structure as of TgRT. Using psi BLAST, we found human thiamine triphosphatase (hTTP) is the only TTM with the available structure that resembles TgRT predicted structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eF). Structural comparison with hTTP revealed that the metal binding sites are identical between the two enzymes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD); however, TgRT lacks the C-terminal plug-in helix, which ensures a topologically closed structure and provides substrate specificity only for thiamine triphosphatase (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eF). Collectively, these results demonstrate the overall similarity in the structure of TgRT with other TTMs. Although the central tunnel structure of TTM is conserved amongst TgRT and hTTP, the substrate recognition and specificity vary between them, and this raises the possibility of development of an inhibitor that specifically targets the entry of RNA substrate into TgRT with minimal to no effect on the hTTP.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA defining feature of eukaryotic gene expression is the addition of an m\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG cap to nascent pre-mRNAs shortly after the initiation of synthesis\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. We aimed to understand the capping process in \u003cem\u003eToxoplasma\u003c/em\u003e to examine parasite-specific variations and to find the contribution of this process to a broader understanding of mRNA metabolism and cellular responses. \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e encodes three separate mRNA capping enzymes: triphosphatase, guanylyltransferase, and cap methyltransferase, similar to that of fungi and distinct from the two enzyme capping systems of metazoans and plants. Biochemical characterization of the triphosphatase TgRT firmly categorized it in the family of metal-dependent phosphohydrolases identified in fungi, DNA viruses, and some protozoa. Despite low similarity with other homologs, guanylyltransferase TgGT and the methyltransferase TgGMT are similar in structure and mechanism to the typical guanylyltransferases and cap methyltransferase enzymes found in all eukaryotes. The capping functions of TgCEs were verified biochemically by testing the productive translation of the in vitro generated capped reporter RNA and genetically through complementation of S. cerevisiae strains lacking endogenous enzymes. The depletion of TgRT resulted in global defects in gene expression and complete arrest of parasite replication (tissue culture and mouse host), highlighting the essential role of triphosphatase in the parasite.\u003c/p\u003e \u003cp\u003eIn eukaryotes, capping is facilitated by direct recruitment of the capping enzymes to the transcription machinery \u003cem\u003evia\u003c/em\u003e interactions with the phosphorylated C-terminal domain (CTD) of Rpb1, the largest subunit of RNAP II\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In many organisms, guanylyltransferase and methyltransferase interact directly with the phosphorylated RNAP II CTD; however, in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e, triphosphatase and in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003epombe\u003c/em\u003e, triphosphatase and guanylyltransferase bind the phosphorylated RNAP II CTD independently\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. These enzymes preferred to bind a CTD substrate that was phosphorylated at Ser5 or doubly phosphorylated at Ser2 and Ser5 in the highly conserved heptapeptide repeats Y\u003csub\u003e1\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eP\u003csub\u003e3\u003c/sub\u003eT\u003csub\u003e4\u003c/sub\u003eS\u003csub\u003e5\u003c/sub\u003eP\u003csub\u003e6\u003c/sub\u003eS\u003csub\u003e7\u003c/sub\u003e. \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e Rbp1-CTD contains a mixture of 10 heptapeptide YSPxSPx sequences instead of conserved YSPTSPS; however, Ser2 and Ser5 residues are conserved in those 10 heptapeptide repeats. The role of Ser5- \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and Ser2-\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003ephosphorylation has been shown in transcription initiation and elongation, respectively, in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e. Hence, it will be interesting to test which \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e capping enzyme interacts with Ser5 and/or Ser2-phosphorylated CTD of Rpb1\u003csup\u003e58\u003c/sup\u003e. Besides, \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e triphosphatase and methyltransferase are exceptionally large proteins with N-terminal extensions, suggesting additional functions or more regulatory roles in coordinating cap formation with RNA synthesis\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Also, it is intriguing to test the distribution of TgRNAP II on chromatin without capping to test whether RNAP II transits to the elongation phase, pauses, or transcribes at a low speed or pausing followed by premature transcription termination\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCap sequencing upon TgRT depletion revealed global gene expression defects, resulting in the complete arrest of parasite replication. The most significantly downregulated transcripts were related to chromatin maintenance, structural integrity, and key kinases, which explains why a halt of parasite relocation occurs even with a transient depletion of TgRT. However, the transcripts related to transporter activities, transferase activity, glucose, and glutamate metabolism were upregulated upon loss of capping. The global transcriptome data (ToxoDB) in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e suggest that in TgRT-depleted parasites, most of these downregulated genes were highly expressed, and the genes showed upregulation expressed at low levels. These findings are consistent with the recent study in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e, where the impact of defective capping (depletion of guanylyltransferase) on genome-wide RNA abundance revealed the reduction of highly expressed mRNAs and the accumulation of lowly expressed and more stable mRNAs\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In yeast, such cap-defective transcripts are detected by an Npl3-mediated surveillance mechanism that triggers decapping and subsequent RNA degradation. In \u003cem\u003eToxoplasma\u003c/em\u003e, the RNA surveillance mechanism and RNA degradation pathways are yet to be studied.\u003c/p\u003e \u003cp\u003eGenerally, glucose and glutamate are the sole physiological nutrient sources in carbon metabolism obligatory for parasite growth and survival\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Hence, unsurprisingly, we observed high transcript levels of carbon metabolism genes in cap-defective parasites. Such transcripts are stabilized probably through a posttranscriptional mechanism that maintains the steady-state mRNA levels of these transcripts for such obligatory parasite function. In such a case, the transcript stabilization could outweigh the lowered level of de novo transcription. Stabilizing such transcripts would eventually limit the decline in the transcription rate and ensure some level of advantage to the parasite to survive under deprivation of either carbon source. The reasons for this stability might be attributed to RNA binding proteins, specificity in the decay pathways, other mRNA modifications, or RNA stability elements such that even in the absence of productive transcription\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, these mechanisms could contribute to maintaining existing mRNA pools of certain genes. This interplay between stabilization and degradation processes is essential for the dynamic regulation of gene expression and might explain the adaptation and survival mechanisms in the parasite. Hence, in the choice to live or die, the parasite prefers to use its energy on basic metabolic processes for its survival than on the process required for its replication, and this explains why DNA replication and packaging genes were downregulated in TgRT-depleted parasites.\u003c/p\u003e \u003cp\u003eThe complete differences between the tertiary structures, active sites, and chemical mechanisms of the RNA triphosphatase component of the mRNA capping system in pathogenic fungi, viruses, and protozoa and those of their metazoan hosts highlight TPase as a target for anti-infective drug discovery\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The triphosphate tunnel metalloenzyme (TTM)-type TPases are essential for the growth of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e (ScCet1)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003epombe\u003c/em\u003e (SpPct1)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, the human-pathogenic fungus \u003cem\u003eAspergillus fumigatuts\u003c/em\u003e (AfTriA)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and the human-pathogenic protozoa \u003cem\u003eT\u003c/em\u003e. \u003cem\u003ebrucei\u003c/em\u003e (TbCet1)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. To our knowledge, it has not been determined whether TTM-type TPase is essential for pathogen growth in the host. Here, we employed an auxin-inducible degron approach to show that \u003cem\u003eToxoplasma\u003c/em\u003e TgRT (TTM-type) is essential for the growth of parasites in both the culture and the mouse host. TgRT showed robust manganese-dependent NTPase activity in an exceptionally wide range of temperatures and pH. The crystal structures of ScCet1\u003csup\u003e23\u003c/sup\u003e and TbCet1\u003csup\u003e37\u003c/sup\u003e revealed that the TTM active site comprises essential amino acids that either coordinate a metal ion or the γ-phosphate or stabilize the tunnel architecture. Most of these active site residues in the triphosphate tunnel are conserved in TgRT, and the alanine mutations of a few of these residues showed a dramatic reduction in the activity, suggesting a similar function as observed for ScCet1\u003csup\u003e67\u003c/sup\u003e and TbCet1\u003csup\u003e37\u003c/sup\u003e in interacting with the 5\u0026prime;-end of the triphosphate RNA substrate. The recent studies on biochemical screening for small-molecule inhibitors showed that TbCet1 could be inhibited using a nanomolar concentration of various classes of phenolic compounds\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, whereas kribellosides, metabolites from actinomycetes, selectively inhibit ScCet1\u003csup\u003e68\u003c/sup\u003e. These inhibitors displayed great potency and selectivity for the target enzyme, indicating separate screening against pathogen-specific TTM-type TPases. Hence, the high-resolution crystal structure of TgRT, along with screening for small-molecule inhibitors, will provide critical insights into the basis for TgRT inhibition. The identified compounds can then be tested for parasite growth inhibition in the culture and mouse (natural \u003cem\u003eToxoplasma\u003c/em\u003e host). It is tempting to speculate that these parasite-specific inhibitors may not inhibit human (host) thiamine triphosphatase (hTTP), the only known TTM with similar metal binding sites\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, owing to the presence of alpha helix at the c-terminal end of hTTP, which positions the thiamine in a way that prevents the entry of non-specific substrates like pppRNA, thereby ensuring specificity\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, our work presents a detailed characterization of three-component RNA-capping machinery in \u003cem\u003eToxoplasma\u003c/em\u003e, where RNA triphosphatase is an essential component with complete divergence in the structure and catalytic mechanism from human ortholog. Lack of capping mediated through RNA triphosphatase depletion results in perturbation of gene expression, which is detrimental to the parasite, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and renders RNA triphosphatase an attractive therapeutic target for \u003cem\u003eToxoplasma\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe institutional ethics committee of National Institute of Animal Biotechnology (IAEC/NIAB/2019/48/ASD) has approved using laboratory animals and research protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial or personal interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKalyani Aswale: Data curation, investigation, analysis and writing manuscript. Abhijit S. Deshmukh: Conceptualization, funding acquisition, investigation and writing manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by an NIAB core grant (C022) for ASD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cap sequencing data for the TgRTmAID-3HA transgenic parasite line upon 8h of treatment with vehicle or IAA has been deposited in NCBI\u0026rsquo;s SRA data with accession numbers SRR27549198 (control) and SRR27560546 (Treatment). The illustration in this study was created using BioRender (https://biorender.com/). The data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is funded by the NIAB core grant (C0022) to ASD. We thank Prof. Stewart Shuman, Sloan Kettering Institute, USA, for yeast mutant strains. KA acknowledges DBT, India, for fellowship.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 30, 1217-1258 (2000).\u003c/li\u003e\n\u003cli\u003eMontoya JG, Liesenfeld O. Toxoplasmosis. Lancet 363, 1965-1976 (2004).\u003c/li\u003e\n\u003cli\u003eCerutti A, Blanchard N, Besteiro S. The Bradyzoite: A Key Developmental Stage for the Persistence and Pathogenesis of Toxoplasmosis. Pathogens 9, (2020).\u003c/li\u003e\n\u003cli\u003eMontazeri M, et al. Drug Resistance in Toxoplasma gondii. Front Microbiol 9, 2587 (2018).\u003c/li\u003e\n\u003cli\u003eRadke JR, Behnke MS, Mackey AJ, Radke JB, Roos DS, White MW. The transcriptome of Toxoplasma gondii. 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Biochim Biophys Acta 1830, 4513-4523 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"apicomplexan parasite, Toxoplasma gondii, mRNA capping, RNA triphosphatase, Cap sequencing, auxin-inducible degron","lastPublishedDoi":"10.21203/rs.3.rs-3875304/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3875304/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe protozoan parasite \u003cem\u003eToxoplasma gondii\u003c/em\u003e is thought to rely on RNA processing to accomplish the differential gene expression needed during life cycle stage transitions. Here, we show how RNA capping, the first major pre-mRNA processing event, safeguards transcript homeostasis in \u003cem\u003eToxoplasma\u003c/em\u003e. A functional RNA capping system of \u003cem\u003eToxoplasma\u003c/em\u003e consists of separate RNA triphosphatase, guanylyltransferase, and guanine-N7-methyltransferase enzymes, which together add 5\u0026rsquo; 7-methylguanosine (m\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG) cap to RNA. The in vitro generated capped RNAs bind to the \u003cem\u003eToxoplasma\u003c/em\u003e translation initiator factor, eIF4E, and are translated to protein in the transfected parasites. Biochemical and genetic characterization demonstrates that among three capping enzymes, triphosphatase (TgRT) is unique and a member of the tunnel family of metal-dependent phosphohydrolases, structurally and mechanistically unrelated to the human cysteine-phosphatase-type RNA triphosphatase. We show that TgRT is essential for pre-mRNA capping and parasite growth through inducible conditional knockdown. TgRT perturbation leads to global diminished m\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eG-capped transcripts, as demonstrated by cap-seq, which resulted in the complete arrest of parasite replication in the culture and the mouse host, protecting them from lethal infection. Overall, this study shows the essential role of TgRT-mediated mRNA capping for parasite survival, thereby presenting RNA triphosphatase as an attractive target for \u003cem\u003eToxoplasma\u003c/em\u003e infection.\u003c/p\u003e","manuscriptTitle":"RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-23 17:53:55","doi":"10.21203/rs.3.rs-3875304/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b87fec9f-4fa9-4949-a2cb-1670b0ef9202","owner":[],"postedDate":"January 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28294593,"name":"Biological sciences/Microbiology/Parasitology/Parasite biology"},{"id":28294594,"name":"Biological sciences/Biochemistry/RNA"}],"tags":[],"updatedAt":"2025-07-02T07:55:06+00:00","versionOfRecord":{"articleIdentity":"rs-3875304","link":"https://doi.org/10.1038/s41467-025-59867-z","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-01 04:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-01-23 17:53:55","video":"","vorDoi":"10.1038/s41467-025-59867-z","vorDoiUrl":"https://doi.org/10.1038/s41467-025-59867-z","workflowStages":[]},"version":"v1","identity":"rs-3875304","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3875304","identity":"rs-3875304","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}