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Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica.

Morf L, Pearson RJ, Wang AS, Singh U - Nucleic Acids Res. (2013)

Bottom Line: Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions.Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies.Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California 94305-5107, USA and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5107, USA.

ABSTRACT
RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5'-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a 'trigger' and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5'-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

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Related in: MedlinePlus

Schematic of siRNA trigger-mediated silencing in E. histolytica. (A) Functional sRNAs are generated to the reporter gene fused to the trigger and the construct is silenced by these sRNAs. sRNAs are lost after plasmid removal. (B) Functional sRNAs are generated to an amebic gene fused to the trigger, which silence the endogenous gene and exogenous constructs with sequence identity to the silenced gene. The sRNAs and gene silencing remain after plasmid removal. (C) No functional sRNAs are generated if a promoter does not drive the trigger fusion construct.
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gkt717-F7: Schematic of siRNA trigger-mediated silencing in E. histolytica. (A) Functional sRNAs are generated to the reporter gene fused to the trigger and the construct is silenced by these sRNAs. sRNAs are lost after plasmid removal. (B) Functional sRNAs are generated to an amebic gene fused to the trigger, which silence the endogenous gene and exogenous constructs with sequence identity to the silenced gene. The sRNAs and gene silencing remain after plasmid removal. (C) No functional sRNAs are generated if a promoter does not drive the trigger fusion construct.

Mentions: Importantly, the trigger-mediated silencing was dependent on the presence of large numbers of AS sRNAs to the trigger region, but independent of the placement of the trigger to a fused gene (5′ or 3′) or to the portion of the gene from which the initial trigger was derived. Interestingly, the same DNA sequence could serve as a trigger in one E. histolytica strain, but not in another strain (if no endogenous sRNAs were present to the trigger sequence). A number of additional important insights into the mechanism of silencing were obtained (outlined in Figure 7). The sRNAs generated by this mechanism have 5′-polyphosphate termini, are 5′ biased, can be generated on nascent transcript, have sequence specificity, require transcription of the trigger-gene fusion and are maintained despite loss of the initial trigger-gene plasmid. Importantly, the generation of functional AS sRNAs occurs regardless of whether the silenced gene has a chromosomal copy (i.e. sRNAs are generated to both luciferase and EhROM1 and both genes are silenced using this mechanism). However, the maintenance of sRNAs after loss of the episomal plasmid is strikingly different: genes with a chromosomal copy have long-term persistence of sRNAs and gene silencing, whereas for genes without a second chromosomal copy (i.e. luciferase) the sRNAs rapidly disappear after removal of the trigger plasmid. The persistence of sRNAs and gene silencing despite loss of the initiating plasmid suggests that the chromosomal locus is able to maintain sRNA production and amplification. Whether this amplified silencing process can be initiated for all genes or can occur from a second episomal copy is not currently known.Figure 7.


Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica.

Morf L, Pearson RJ, Wang AS, Singh U - Nucleic Acids Res. (2013)

Schematic of siRNA trigger-mediated silencing in E. histolytica. (A) Functional sRNAs are generated to the reporter gene fused to the trigger and the construct is silenced by these sRNAs. sRNAs are lost after plasmid removal. (B) Functional sRNAs are generated to an amebic gene fused to the trigger, which silence the endogenous gene and exogenous constructs with sequence identity to the silenced gene. The sRNAs and gene silencing remain after plasmid removal. (C) No functional sRNAs are generated if a promoter does not drive the trigger fusion construct.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814356&req=5

gkt717-F7: Schematic of siRNA trigger-mediated silencing in E. histolytica. (A) Functional sRNAs are generated to the reporter gene fused to the trigger and the construct is silenced by these sRNAs. sRNAs are lost after plasmid removal. (B) Functional sRNAs are generated to an amebic gene fused to the trigger, which silence the endogenous gene and exogenous constructs with sequence identity to the silenced gene. The sRNAs and gene silencing remain after plasmid removal. (C) No functional sRNAs are generated if a promoter does not drive the trigger fusion construct.
Mentions: Importantly, the trigger-mediated silencing was dependent on the presence of large numbers of AS sRNAs to the trigger region, but independent of the placement of the trigger to a fused gene (5′ or 3′) or to the portion of the gene from which the initial trigger was derived. Interestingly, the same DNA sequence could serve as a trigger in one E. histolytica strain, but not in another strain (if no endogenous sRNAs were present to the trigger sequence). A number of additional important insights into the mechanism of silencing were obtained (outlined in Figure 7). The sRNAs generated by this mechanism have 5′-polyphosphate termini, are 5′ biased, can be generated on nascent transcript, have sequence specificity, require transcription of the trigger-gene fusion and are maintained despite loss of the initial trigger-gene plasmid. Importantly, the generation of functional AS sRNAs occurs regardless of whether the silenced gene has a chromosomal copy (i.e. sRNAs are generated to both luciferase and EhROM1 and both genes are silenced using this mechanism). However, the maintenance of sRNAs after loss of the episomal plasmid is strikingly different: genes with a chromosomal copy have long-term persistence of sRNAs and gene silencing, whereas for genes without a second chromosomal copy (i.e. luciferase) the sRNAs rapidly disappear after removal of the trigger plasmid. The persistence of sRNAs and gene silencing despite loss of the initiating plasmid suggests that the chromosomal locus is able to maintain sRNA production and amplification. Whether this amplified silencing process can be initiated for all genes or can occur from a second episomal copy is not currently known.Figure 7.

Bottom Line: Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions.Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies.Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California 94305-5107, USA and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5107, USA.

ABSTRACT
RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5'-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a 'trigger' and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5'-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

Show MeSH
Related in: MedlinePlus