High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress

Zhang, Hanbang; Ehrenkaufer, Gretchen; Manna, Dipak; Hall, Neil; Singh, Upinder

doi:10.1371/journal.pone.0134481

Cited by 12 publications

(49 citation statements)

References 44 publications

(93 reference statements)

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“…Gene silencing occurs due to the generation of antisense sRNAs to the fused gene. Our recent work with sequencing the sRNA repertoire in E. invadens afforded us the opportunity to adapt the trigger method for use in E. invadens (41). In order to identify genes silenced endogenously by the RNAi pathway in E. invadens, we characterized the sRNA libraries from trophozoites and early-cyst (24-hour encystation), mature-cyst (72-hour encystation), and excysting (8 hours after induction of excystation) parasites.…”

Section: Resultsmentioning

confidence: 99%

“…mRNA transcript expression (37,41). However, unlike E. histolytica, the E. invadens promoter regions had a greater number of sRNAs mapping to them, and there was a wider distribution of both sense (S) and antisense sRNAs over the length of a given gene's coding region.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, it is imperative to determine if silencing is stable during this massive change in cell physiology. Given that the E. invadens sRNA profile remains unchanged during the interconversion between trophozoites and cysts, we presume that the silencing will persist (41). To determine if trigger-mediated gene silencing is stable under both encystation and excystation conditions, the cell lines 003-Trigger-Luc and 152-Trigger-Luc were encysted, and the firefly luciferase activity was measured at two different time points: 24 hours (early cysts) and 72 hours (mature cysts).…”

Section: Resultsmentioning

confidence: 99%

“…The sRNAs have 5= polyphosphate termini and are similar to secondary sRNAs generated in nematodes (39,40). The genome of E. invadens reveals the presence of several genes that code for the RNAi machinery, including four genes that encode full or partial Argonaute proteins, two genes that encode RNA-dependent RNA polymerase (RdRP), and a gene with an RNase III domain-containing protein (41). The presence of these genes indicates that the RNAi pathway may be functional in E. invadens.…”

mentioning

confidence: 98%

“…The presence of these genes indicates that the RNAi pathway may be functional in E. invadens. Furthermore, recent work has identified the presence of ϳ27-nt sRNAs in E. invadens with features similar to those of E. histolytica sRNAs, including 5= polyphosphate (polyP) termini and an association of 27-nt sRNAs with silenced genes (41).…”

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confidence: 99%

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Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

et al. 2016

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b Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5= or 3= end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 98%

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confidence: 99%

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Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

et al. 2016

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Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica

Khalil

Foda

Suresh

et al. 2016

International Journal for Parasitology

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Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a “trigger” to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.

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Rab GTPases take centre stage in understandingEntamoeba histolyticabiology

2018

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Rab GTPases constitute the largest subgroup in the Ras superfamily of GTPases. It is well established that different Rab GTPases are localized in discrete subcellular localization and regulate the membrane trafficking in nearly all eukaryotic cells. Rab GTPase diversity is often regarded as an expression of vesicular trafficking complexity. The pathogenic amoeba Entamoeba histolytica harbours 91 Rab GTPases which is the highest among the currently available genome sequences from the eukaryotic kingdom. Here, we review the current status of amoebic Rab GTPases diversity, unique biochemical and structural features and summarise their predicted regulators. We discuss how amoebic Rab GTPases are involved in cellular processes such as endocytosis, phagocytosis, and invasion of host cellular components, which are essential for parasite survival and virulence.

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High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress

Cited by 12 publications

References 44 publications

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica

Rab GTPases take centre stage in understandingEntamoeba histolyticabiology

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