Characterization of Toxoplasma gondii Spt5 like transcription elongation factor

Mitra, Pallabi; Deshmukh, Abhijit; Gurupwar, Rajkumar; Kashyap, Poonam

doi:10.1016/j.bbagrm.2019.01.003

Cited by 9 publications

(5 citation statements)

References 75 publications

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“…The inhibition of CDK7 kinase causes transcript dysregulation in T. gondii [33]. P Mitra, et al [34] reported that T. gondii CRK9 regulates RNA polymerase II by phosphorylating the T. gondii ribo avin binding protein 1 carboxyl-terminal domain. The loss of cytoplasmic TgCRK2 results in T. gondii cell cycle G1 phase arrest [14].…”

Section: Discussionmentioning

confidence: 99%

Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

Zhou

Ding

Yang

et al. 2020

Preprint

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Background：Coccidiosis caused by Eimeria tenella infection, directly or indirectly leads to great loss to poultry industry. With the emergence of drug-resistance in chicken coccidia, it is imperative to develop new drugs. Cyclin-dependent kinases (CDKs) regulate cell cycle progression in numerous organisms by acting as key molecular switches. Results: In the present study, a diclazuril anticoccidiosis animal model was established and CDK-related kinase 2 (CRK2) in the second-generation merozoite of E. tenella (EtCRK2) gene was amplified through reverse transcription-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli Rosetta (DE3). Purified recombinant protein was used for antiserum preparation. Subsequently, EtCRK2 transcription and translation levels were detected through quantitative real-time PCR and Western blot analysis, respectively. The localization of EtCRK2 in merozoites was examined via immunofluorescence techniques. Results showed that the mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localized in the cytoplasm of merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. Conclusions: This study demonstrated that the anticoccidial drug diclazuril against E. tenella by affecting the expression pattern of EtCRK2 molecule, and EtCRK2 may be used as a candidate target for new drug development.

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Section: Discussionmentioning

confidence: 99%

Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

Zhou

Ding

Yang

et al. 2020

Preprint

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“…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 NdeI-EcoRI 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 Nterminal (TgRT, TgGT, and TgGMT) and C-terminal (TgeIF4E) 6-xHis-tag and puri ed on a nickel-nitrilotriacetic acid-agarose resin column as described previously 29 . Brie y, 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.…”

Section: Parasite Culturementioning

confidence: 99%

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

Deshmukh,

Aswale

2024

Preprint

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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 (m7G) 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 m7G-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.

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“…Metazoan ELF1 is a conserved transcription elongation factor that coordinates with Spt4-Spt5 in yeast or DSIF in humans to facilitate RNAPII-mediated transcription [7][8][9]. Following our recent identification of Spt4 and Spt5 in Toxoplasma [16], we were interested in looking up the ELF1 homolog. BLASTP search of the Toxoplasma database (http://toxodb.org/toxo/) was undertaken to identify the Toxoplasma counterpart of ELF1 using amino acid sequences of yeast and human ELF1 as a query.…”

Section: Identification and In Silico Analysis Of Tgme49_297730mentioning

confidence: 99%

“…Similarly, polyclonal antibodies to recombinant TgBAG1 were raised in-house for this study. TgCST1, TgSAG1, and TgIMC1 antibodies were used from previous studies [15,16].…”

Section: Antibodiesmentioning

confidence: 99%

“…Yeast mutant strains (DELF1DDST, FY2376) were transformed with a plasmid carrying TgELD and selected in the presence and absence of 6 Aza uracil (6-AU) on SD-TRP plates. Western blot analysis was performed as described previously [16] to check for the expression of the complemented genes in respective transformants using an anti-His antibody. ScGAPDH expression levels were used as a control.…”

Section: Yeast Complementationmentioning

confidence: 99%

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A functionally divergent transcription elongation factor 1‐like protein in Toxoplasma gondii

et al. 2021

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Zinc ribbons, one of the largest fold groups among zinc fingers, often include proteins involved in the transcription machinery. Here, we identify and characterize one such zinc ribbon-bearing protein in the apicomplexan parasite Toxoplasma gondii, annotated as putative transcription elongation factor 1 (ELF1), with predicted functions in transcription and chromatin maintenance. We show that this ELF1 homolog, referred to as T. gondii ELF1-like divergent (TgELD), is expressed in both tachyzoite and bradyzoite developmental stages. TgELD associates with the cytoskeleton in the tachyzoites, while it transiently becomes a part of the cyst wall in the early bradyzoites, followed by a cytosolic and peripheral localization in late bradyzoites. TgELD is phosphorylated by a casein kinase 2-like protein, which has potential implications for its localization and function in the parasite.

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Characterization of Toxoplasma gondii Spt5 like transcription elongation factor

Cited by 9 publications

References 75 publications

Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

Diclazuril-Induced Expression of CDK-Related Kinase 2 in the Second-Generation Merozoites of Eimeria Tenella

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

A functionally divergent transcription elongation factor 1‐like protein in Toxoplasma gondii

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