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Updated: Feb 23, 2026
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Published on: July 12, 2013
mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii
Margaret A Nakamoto1, Alexander F Lovejoy1, Alicja M Cygan1
1Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.
Abstract:
RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3'-UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.
Insights
Pseudouridine (Ψ), an abundant RNA modification, plays a crucial role in Toxoplasma gondii differentiation. This study identifies Ψ sites and reveals their impact on mRNA stability and gene expression, suggesting a significant biological function.
Area of Science:
- Molecular Biology
- RNA Biology
- Parasitology
Background:
- Over 100 post-transcriptional RNA modifications exist, with pseudouridine (Ψ) being abundant but its biological role not fully understood.
- Pseudouridine synthase (TgPUS1) is essential for differentiation in the parasite *Toxoplasma gondii*.
- Understanding pseudouridylation's role is key to deciphering RNA regulation and parasite biology.
Purpose of the Study:
- To develop methods for identifying TgPUS1-dependent pseudouridylation sites in *Toxoplasma* RNA.
- To investigate the impact of pseudouridylation on mRNA stability and gene expression using TgPUS1 mutants.
- To elucidate the biological significance of pseudouridylation in *Toxoplasma* development.
Main Methods:
- Gel-based and deep-sequencing techniques to map pseudouridylation sites.
- Comparative RNA sequencing (RNA-seq) of wild-type and TgPUS1-mutant *Toxoplasma*.
- Pulse/chase labeling with 4-thiouracil to assess mRNA half-life.
Main Results:
- Identified conserved pseudouridylation in rRNA, tRNA, and snRNA, and extensive pseudouridylation in mRNA.
- TgPUS1-dependent pseudouridines were found enriched at codon position 1 in mRNAs and depleted in 3'-UTRs.
- TgPUS1-dependent pseudouridylation affects developmentally regulated transcripts, increasing mRNA abundance and half-life in mutant parasites.
Conclusions:
- Pseudouridylation, mediated by TgPUS1, significantly impacts mRNA stability and gene expression in *Toxoplasma*.
- This modification is crucial for regulating developmentally important transcripts.
- The findings provide initial evidence for a substantial biological role of mRNA pseudouridylation.
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