Dataset Information

Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii.

ABSTRACT: Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii.Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity.TgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif.TgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.

SUBMITTER: Liu M

PROVIDER: S-EPMC3997814 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii.

Liu Min M Miao Jun J Liu Tingkai T Sullivan William J WJ Cui Liwang L Chen Xiaoguang X

Parasites & vectors 20140331

<h4>Background</h4>Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii.<h4>Methods</h4>Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgP ...[more]

PMID: 24685055

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Project description:BACKGROUND: Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. RESULTS: The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. CONCLUSIONS: The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.

Project description:Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+ Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite's rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion.IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite's infection cycle.

Project description:Reprogramming of a gene's expression pattern by acquisition and loss of sequences recognized by specific regulatory RNA binding proteins may be a major mechanism in the evolution of biological regulatory programs. We identified that RNA targets of Puf3 orthologs have been conserved over 100-500 million years of evolution in five eukaryotic lineages. Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious model for their evolutionary history. This model entails extensive and coordinated changes in the Puf targets as well as changes in the number of Puf genes and alterations of RNA binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein products are predominantly involved in the production and organization of mitochondrial complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one lineage maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regulator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in budding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 target set in filamentous fungi expanded to include mitochondrial genes involved in the tricarboxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not bound by Puf3 in budding yeast, observations that provide additional evidence for substantial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its targets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial electron transport chain (ETC) complex I as well as hundreds of other mRNAs with nonmitochondrial functions. The many concerted and conserved changes in the RNA targets of Puf proteins strongly support an extensive role of RNA binding proteins in coordinating gene expression, as originally proposed by Keene. Rewiring of Puf-coordinated mRNA targets and transcriptional control of the same genes occurred at different points in evolution, suggesting that there have been distinct adaptations via RNA binding proteins and transcription factors. The changes in Puf targets and in the Puf proteins indicate an integral involvement of RNA binding proteins and their RNA targets in the adaptation, reprogramming, and function of gene expression.

Dataset Information

Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii.

Publications

Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii.

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