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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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Gene knockdown of EhMyb1 reveals that AS sRNAs are generated to nascent transcript. (A) N-terminal Myc-tagged EhMyb has nuclear localization. (B) E. histolytica HM-1:IMSS parasites stably transfected with the plasmid containing the EHI_197520 trigger fused to the EhMyb1 gene (cell line: 19-trigger-Myb). RT-PCR for EhMyb1 in the 19-trigger-Myb cell line demonstrates the absence of EhMyb mRNA in the 19-trigger-Myb cell line. Control (EHI_199600) indicates equivalent mRNA in both lanes. (C) Northern blot of the 19-trigger-Myb cell line probed for EhMyb1 AS sRNAs reveals an abundant population of AS sRNA to the EhMyb1 gene. AS sRNAs that map to the intron of EhMyb1 were detected. Control (EHI_118130) indicates equivalent sRNA in both lanes (75 µg of enriched sRNA). (D) Viability assay of stress-treated parasites (1 h, 1 mM H2O2) revealed that EhMyb1 knockdown parasites had significantly decreased viability in response to stress.
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gkt717-F6: Gene knockdown of EhMyb1 reveals that AS sRNAs are generated to nascent transcript. (A) N-terminal Myc-tagged EhMyb has nuclear localization. (B) E. histolytica HM-1:IMSS parasites stably transfected with the plasmid containing the EHI_197520 trigger fused to the EhMyb1 gene (cell line: 19-trigger-Myb). RT-PCR for EhMyb1 in the 19-trigger-Myb cell line demonstrates the absence of EhMyb mRNA in the 19-trigger-Myb cell line. Control (EHI_199600) indicates equivalent mRNA in both lanes. (C) Northern blot of the 19-trigger-Myb cell line probed for EhMyb1 AS sRNAs reveals an abundant population of AS sRNA to the EhMyb1 gene. AS sRNAs that map to the intron of EhMyb1 were detected. Control (EHI_118130) indicates equivalent sRNA in both lanes (75 µg of enriched sRNA). (D) Viability assay of stress-treated parasites (1 h, 1 mM H2O2) revealed that EhMyb1 knockdown parasites had significantly decreased viability in response to stress.

Mentions: To characterize EhMyb1 (EHI_197980), a putative transcription factor that was upregulated during H2O2 stress (32), we tagged the protein with an N-terminal Myc tag. Protein localization to the parasite nucleus supports the hypothesis that EhMyb1 is a transcription factor (Figure 6A). We designed a 19-trigger-EhMyb1 construct and achieved robust gene knockdown in the E. histolytica HM-1:IMSS strain; silencing was associated with generation of an abundant population of sRNAs to the EhMyb1 gene (Figure 6B and C). As the EhMyb1 gene has an intron, we probed for AS sRNAs to the intronic region and found that they could be detected, indicating that the AS sRNAs can be generated from nascent transcript. This is similar to data from sequencing endogenous sRNA populations, where sRNAs were detected to introns as well as exon–exon junctions (16,38). To determine the relative abundance of AS sRNAs along the gene, we probed for AS sRNAs at discrete locations and observed that AS sRNAs were most abundant at the 5′ portion of the gene, with a decrease in the 3′ portion of the gene (data not shown). We also probed for sense sRNAs but could not detect them (data not shown). Overall, the sRNAs generated by the trigger-gene fusion (abundant AS sRNAs biased to the 5′ end of a gene with 5′-polyphosphate termini and no detectable sense sRNAs) are highly similar to the endogenous 27-nt sRNA population in E. histolytica (14).Figure 6.


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)

Gene knockdown of EhMyb1 reveals that AS sRNAs are generated to nascent transcript. (A) N-terminal Myc-tagged EhMyb has nuclear localization. (B) E. histolytica HM-1:IMSS parasites stably transfected with the plasmid containing the EHI_197520 trigger fused to the EhMyb1 gene (cell line: 19-trigger-Myb). RT-PCR for EhMyb1 in the 19-trigger-Myb cell line demonstrates the absence of EhMyb mRNA in the 19-trigger-Myb cell line. Control (EHI_199600) indicates equivalent mRNA in both lanes. (C) Northern blot of the 19-trigger-Myb cell line probed for EhMyb1 AS sRNAs reveals an abundant population of AS sRNA to the EhMyb1 gene. AS sRNAs that map to the intron of EhMyb1 were detected. Control (EHI_118130) indicates equivalent sRNA in both lanes (75 µg of enriched sRNA). (D) Viability assay of stress-treated parasites (1 h, 1 mM H2O2) revealed that EhMyb1 knockdown parasites had significantly decreased viability in response to stress.
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gkt717-F6: Gene knockdown of EhMyb1 reveals that AS sRNAs are generated to nascent transcript. (A) N-terminal Myc-tagged EhMyb has nuclear localization. (B) E. histolytica HM-1:IMSS parasites stably transfected with the plasmid containing the EHI_197520 trigger fused to the EhMyb1 gene (cell line: 19-trigger-Myb). RT-PCR for EhMyb1 in the 19-trigger-Myb cell line demonstrates the absence of EhMyb mRNA in the 19-trigger-Myb cell line. Control (EHI_199600) indicates equivalent mRNA in both lanes. (C) Northern blot of the 19-trigger-Myb cell line probed for EhMyb1 AS sRNAs reveals an abundant population of AS sRNA to the EhMyb1 gene. AS sRNAs that map to the intron of EhMyb1 were detected. Control (EHI_118130) indicates equivalent sRNA in both lanes (75 µg of enriched sRNA). (D) Viability assay of stress-treated parasites (1 h, 1 mM H2O2) revealed that EhMyb1 knockdown parasites had significantly decreased viability in response to stress.
Mentions: To characterize EhMyb1 (EHI_197980), a putative transcription factor that was upregulated during H2O2 stress (32), we tagged the protein with an N-terminal Myc tag. Protein localization to the parasite nucleus supports the hypothesis that EhMyb1 is a transcription factor (Figure 6A). We designed a 19-trigger-EhMyb1 construct and achieved robust gene knockdown in the E. histolytica HM-1:IMSS strain; silencing was associated with generation of an abundant population of sRNAs to the EhMyb1 gene (Figure 6B and C). As the EhMyb1 gene has an intron, we probed for AS sRNAs to the intronic region and found that they could be detected, indicating that the AS sRNAs can be generated from nascent transcript. This is similar to data from sequencing endogenous sRNA populations, where sRNAs were detected to introns as well as exon–exon junctions (16,38). To determine the relative abundance of AS sRNAs along the gene, we probed for AS sRNAs at discrete locations and observed that AS sRNAs were most abundant at the 5′ portion of the gene, with a decrease in the 3′ portion of the gene (data not shown). We also probed for sense sRNAs but could not detect them (data not shown). Overall, the sRNAs generated by the trigger-gene fusion (abundant AS sRNAs biased to the 5′ end of a gene with 5′-polyphosphate termini and no detectable sense sRNAs) are highly similar to the endogenous 27-nt sRNA population in E. histolytica (14).Figure 6.

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