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High-dose siRNAs upregulate mouse Eri-1 at both transcription and posttranscription levels.

Bian Y, Zhou W, Zhao Y, Li X, Geng W, Hao R, Yang Q, Huang W - PLoS ONE (2011)

Bottom Line: High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency.In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1.Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Life Science, Fudan University, Shanghai, China.

ABSTRACT
The eri-1 gene encodes a 3' exonuclease that can negatively regulate RNA interference via siRNase activity. High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency. Here we report that hd-siRNAs induce mouse Eri-1 (meri-1) expression through the recruitment of Sp1, Ets-1, and STAT3 to the meri-1 promoter and the formation of an Sp1-Ets-1-STAT3 complex. In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1. Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

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Sp1 and Ets-1 binding sites are the key cis-elements for the siRNA-stimulated meri-1 promoter activity.The hd-siRNA-stimulated transcriptional activity of the truncated meri-1 promoters was determined using the GFP reporter assay and the secreted alkaline phosphatase (SEAP) activity assay. (A) GFP assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with Nc siRNA_1 and GFP reporter constructs containing the truncated meri-1 promoters (I–VI). (B) SEAP activity assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1. (C) Upper panels: A schematic presentation of the putative trans-factor binding sites in the −87 to −28 region of the meri-1 promoter, as predicted by MAPPER [48]. Middle panels: The SEAP activity in the culture media of the CHO cells co-transfected with Nc siRNA_1 and the SEAP reporter constructs, including the 180-bp meri-1 promoter (Peri-1(−87)) (panel 1), the 160-bp meri-1 promoter with deletions of Sp1 and NRF-1 site (Peri-1(−67)) (panel 2), the same fragment with a mutation in the Ets-1 binding site (Peri-1(−67)mEts-1) (panel 3), the same sequence as Peri-1(−47) but with the Sp1 site added to the 5′ end (Peri-1(−84Δ−70–−48)) (panel 4), the same sequence as Peri-1(−27) but with the Sp1 site added at the 5′ end (Peri-1(−84Δ−70–−30)) (panel 5), or the 120-bp meri-1 promoter (Peri-1(−27)) (panel 6). Lower panel: The SEAP activity in the culture media of the CHO cells transfected with the truncated meri-1 promoters. The CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1 or 21-bp DNA control. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples.
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pone-0026466-g002: Sp1 and Ets-1 binding sites are the key cis-elements for the siRNA-stimulated meri-1 promoter activity.The hd-siRNA-stimulated transcriptional activity of the truncated meri-1 promoters was determined using the GFP reporter assay and the secreted alkaline phosphatase (SEAP) activity assay. (A) GFP assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with Nc siRNA_1 and GFP reporter constructs containing the truncated meri-1 promoters (I–VI). (B) SEAP activity assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1. (C) Upper panels: A schematic presentation of the putative trans-factor binding sites in the −87 to −28 region of the meri-1 promoter, as predicted by MAPPER [48]. Middle panels: The SEAP activity in the culture media of the CHO cells co-transfected with Nc siRNA_1 and the SEAP reporter constructs, including the 180-bp meri-1 promoter (Peri-1(−87)) (panel 1), the 160-bp meri-1 promoter with deletions of Sp1 and NRF-1 site (Peri-1(−67)) (panel 2), the same fragment with a mutation in the Ets-1 binding site (Peri-1(−67)mEts-1) (panel 3), the same sequence as Peri-1(−47) but with the Sp1 site added to the 5′ end (Peri-1(−84Δ−70–−48)) (panel 4), the same sequence as Peri-1(−27) but with the Sp1 site added at the 5′ end (Peri-1(−84Δ−70–−30)) (panel 5), or the 120-bp meri-1 promoter (Peri-1(−27)) (panel 6). Lower panel: The SEAP activity in the culture media of the CHO cells transfected with the truncated meri-1 promoters. The CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1 or 21-bp DNA control. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples.

Mentions: We analyzed the meri-1 promoter for potential hd-siRNA responsive elements as follows. Several GFP or SEAP reporter constructs driven by meri-1 promoter fragments of various lengths were made and transfected into CHO cells. Similar reporter activity (<10% difference) upon siRNA treatment was observed for the 1.8 kb promoter (Peri-1(−1654)), the 500 bp (Peri-1(−386)), and the 200 bp fragments (Peri-1(−87)) (Figure 2A: I–III; Figure 2B: panels 1–3). However, the 140 bp fragment (Peri-1(−27)) lost most of the promoter activity (Figure 2A: IV; Figure 2B: panel 7), indicating that −87 to −27 region contains critical elements responsible for hd-siRNA stimulated meri-1 expression.


High-dose siRNAs upregulate mouse Eri-1 at both transcription and posttranscription levels.

Bian Y, Zhou W, Zhao Y, Li X, Geng W, Hao R, Yang Q, Huang W - PLoS ONE (2011)

Sp1 and Ets-1 binding sites are the key cis-elements for the siRNA-stimulated meri-1 promoter activity.The hd-siRNA-stimulated transcriptional activity of the truncated meri-1 promoters was determined using the GFP reporter assay and the secreted alkaline phosphatase (SEAP) activity assay. (A) GFP assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with Nc siRNA_1 and GFP reporter constructs containing the truncated meri-1 promoters (I–VI). (B) SEAP activity assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1. (C) Upper panels: A schematic presentation of the putative trans-factor binding sites in the −87 to −28 region of the meri-1 promoter, as predicted by MAPPER [48]. Middle panels: The SEAP activity in the culture media of the CHO cells co-transfected with Nc siRNA_1 and the SEAP reporter constructs, including the 180-bp meri-1 promoter (Peri-1(−87)) (panel 1), the 160-bp meri-1 promoter with deletions of Sp1 and NRF-1 site (Peri-1(−67)) (panel 2), the same fragment with a mutation in the Ets-1 binding site (Peri-1(−67)mEts-1) (panel 3), the same sequence as Peri-1(−47) but with the Sp1 site added to the 5′ end (Peri-1(−84Δ−70–−48)) (panel 4), the same sequence as Peri-1(−27) but with the Sp1 site added at the 5′ end (Peri-1(−84Δ−70–−30)) (panel 5), or the 120-bp meri-1 promoter (Peri-1(−27)) (panel 6). Lower panel: The SEAP activity in the culture media of the CHO cells transfected with the truncated meri-1 promoters. The CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1 or 21-bp DNA control. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198429&req=5

pone-0026466-g002: Sp1 and Ets-1 binding sites are the key cis-elements for the siRNA-stimulated meri-1 promoter activity.The hd-siRNA-stimulated transcriptional activity of the truncated meri-1 promoters was determined using the GFP reporter assay and the secreted alkaline phosphatase (SEAP) activity assay. (A) GFP assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with Nc siRNA_1 and GFP reporter constructs containing the truncated meri-1 promoters (I–VI). (B) SEAP activity assay testing the activity of the truncated meri-1 promoters. CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1. (C) Upper panels: A schematic presentation of the putative trans-factor binding sites in the −87 to −28 region of the meri-1 promoter, as predicted by MAPPER [48]. Middle panels: The SEAP activity in the culture media of the CHO cells co-transfected with Nc siRNA_1 and the SEAP reporter constructs, including the 180-bp meri-1 promoter (Peri-1(−87)) (panel 1), the 160-bp meri-1 promoter with deletions of Sp1 and NRF-1 site (Peri-1(−67)) (panel 2), the same fragment with a mutation in the Ets-1 binding site (Peri-1(−67)mEts-1) (panel 3), the same sequence as Peri-1(−47) but with the Sp1 site added to the 5′ end (Peri-1(−84Δ−70–−48)) (panel 4), the same sequence as Peri-1(−27) but with the Sp1 site added at the 5′ end (Peri-1(−84Δ−70–−30)) (panel 5), or the 120-bp meri-1 promoter (Peri-1(−27)) (panel 6). Lower panel: The SEAP activity in the culture media of the CHO cells transfected with the truncated meri-1 promoters. The CHO cells were co-transfected with SEAP reporter constructs containing the truncated meri-1 promoters and Nc siRNA_1 or 21-bp DNA control. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples.
Mentions: We analyzed the meri-1 promoter for potential hd-siRNA responsive elements as follows. Several GFP or SEAP reporter constructs driven by meri-1 promoter fragments of various lengths were made and transfected into CHO cells. Similar reporter activity (<10% difference) upon siRNA treatment was observed for the 1.8 kb promoter (Peri-1(−1654)), the 500 bp (Peri-1(−386)), and the 200 bp fragments (Peri-1(−87)) (Figure 2A: I–III; Figure 2B: panels 1–3). However, the 140 bp fragment (Peri-1(−27)) lost most of the promoter activity (Figure 2A: IV; Figure 2B: panel 7), indicating that −87 to −27 region contains critical elements responsible for hd-siRNA stimulated meri-1 expression.

Bottom Line: High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency.In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1.Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Life Science, Fudan University, Shanghai, China.

ABSTRACT
The eri-1 gene encodes a 3' exonuclease that can negatively regulate RNA interference via siRNase activity. High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency. Here we report that hd-siRNAs induce mouse Eri-1 (meri-1) expression through the recruitment of Sp1, Ets-1, and STAT3 to the meri-1 promoter and the formation of an Sp1-Ets-1-STAT3 complex. In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1. Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

Show MeSH