tRNA Gene Sequences Are Required for Transcriptional Silencing in Entamoeba histolytica

Abstract

Transcriptional silencing by trans inactivation can contribute to the regulation of gene expression in eukaryotic cells. In the human intestinal protozoan parasite Entamoeba histolytica, trans inactivation of the amoebapore-A gene (AP-A) was recently achieved by episomal transfection of E. histolytica trophozoites with the plasmid psAP1. The mechanism of AP-A trans inactivation is largely unknown, though it was suggested that a partial short interspersed transposable element (SINE) is required. By systematic assessment of various E. histolytica isolates transfected with psAP1 derivates, trans inactivation of AP-A was restricted to the strain HM-1:IMSS (2411) but could not be achieved in other standard laboratory strains. Importantly, sequences of an E. histolytica tRNA array that were located on psAP1 in close proximity to the AP-A upstream region and comprising the glutamic acid (TTC) (E) and tyrosine (GTA) (Y) tRNA genes were indispensable for AP-A silencing. In contrast to the case described in previous reports, SINE was not required for AP-A trans inactivation. AP-A expression could be regained in silenced cells by episomal transfection under the control of a heterologous E. histolytica promoter, opening a way toward future silencing of individual genes of interest in E. histolytica. Our results indicate that tRNA gene-mediated silencing is not restricted to Saccharomyces cerevisiae.

RNA Interference In Entamoeba Histolytica : Implications For Parasite Biology And Gene Silencing

“…While the above data may point to a transcriptional silencing mechanism linked with long non-coding RNAs and histone modification, a different mechanism, trans -inactivation through tRNA gene-mediated silencing, was recently reported to be linked to the observed AP-A silencing [69]. In this study, sequence comparison between plasmid pNAP-A (that caused AP-A overexpression) and plasmid psAP1 (that caused AP-A silencing) revealed some sequence differences.…”

“…The psAP1 contains an extended actin 3′ sequence, where a tRNA array comprising the glutamic acid (TTC) and tyrosine (GTA) tRNAs, resides. Testing with a series of 5′ and 3′ deletion constructs showed the 3′ end tRNA genes are sufficient to mediate AP-A trans -inactivation, while the partial SINE1 element and the T-rich region located at the 5′ flanking region of AP-A seemed to be dispensable [69]. …”

Epigenetics in The Unicellular Parasite Entamoeba Histolytica

“…The exact mechanism that underlies this silencing is currently unknown. The silencing occurs only in a specific E. histolytica strain [57]. In addition, dsRNA, DNA methylation and DNA rearrangements are not involved in this silencing.…”

“…On the other hand, it has been reported recently that the sequences that encode tyrosine and glutamic acid tRNAs genes, which are located upstream to the amoebapore gene promoter are essential for the induction of silencing [57]. The effect that tRNA genes have on the transcription of neighboring genes is referred as tRNA gene-mediated silencing.…”

Virulence and virulence factors in Entamoeba histolytica, the agent of human amoebiasis

“…Interestingly, irreversible silencing of amoebapore (AP), a pore-forming protein believed to be involved in pathogenicity, in E. histolytica was obtained following transfection with a plasmid containing sequences of a SINE [26]. Further work suggests that the presence of two tRNA genes is responsible for this unusual epigenetic phenomenon [27]. These observations led to speculate that transposable elements inserted in the neighbourhood of genes could lead to the increased gene expression related to virulence [14].…”