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Postsplicing-Derived Full-Length Intron Circles in the Protozoan Parasite Entamoeba histolytica.

ABSTRACT: Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5'ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5'ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5'-3'ss ligation points. Different from the reported functions of ciRNAs, the 5'ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.

SUBMITTER: Mendoza-Figueroa MS

PROVIDER: S-EPMC6085484 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Postsplicing-Derived Full-Length Intron Circles in the Protozoan Parasite <i>Entamoeba histolytica</i>.

Mendoza-Figueroa María S MS Alfonso-Maqueira Eddy E EE Vélez Cristina C Azuara-Liceaga Elisa I EI Zárate Selene S Villegas-Sepúlveda Nicolás N Saucedo-Cárdenas Odila O Valdés Jesús J

Frontiers in cellular and infection microbiology 20180803

Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. <i>Entamoeba histolytica</i>, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic ...[more]

PMID: 30123775

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Project description:BackgroundHistone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known.ResultsIn order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)).ConclusionThis work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

Project description:PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

Dataset Information

Postsplicing-Derived Full-Length Intron Circles in the Protozoan Parasite Entamoeba histolytica.

Publications

Postsplicing-Derived Full-Length Intron Circles in the Protozoan Parasite <i>Entamoeba histolytica</i>.

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