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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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Involvement of Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) Knockdown of Sp1 and Ets-1 genes inhibits the siRNA-stimulated meri-1 promoter activity. A: The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus siSp1_1, siSp1_2, siEts-1_1 or siEts-1_2 was measured. The knockdown effects were shown in lower panel. (B) Overexpression of the Sp1 and Ets-1 dominant-negative mutant inhibits the siRNA-stimulated meri-1 promoter activity. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus pCMV-HA, pCMV-HA-Sp1(DN) or pCMV-HA-Ets-1(DN) was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C)–(F) Sp1 and Ets-1 binding specifically to the meri-1 promoter. (C) and (D) EMSAs were performed using various biotin-labeled oligonucleotides corresponding to the putative Sp1 and Ets-1 elements in the meri-1 promoter, as described in Table 1. Nuclear extracts were prepared from CHO cells pre-treated with Nc siRNA_1. The competition assays were performed with or without a 50-fold excess of each unlabelled oligonucleotide, as indicated above each lane. Immunodepletion EMSAs were performed using 1 µl of the anti-Sp1, anti-Ets-1 antibodies and control normal mouse IgG, as indicated above each lane. Immunodepletion effects were shown under the related lanes. (E) Cell extracts of the CHO cells pre-treated with or not with Nc siRNA were prepared. The aliquots were subjected to DNA affinity precipitation assay (DAPA) with biotinylated oligonucleotides that contained the Sp1-, Ets-1- or both Sp1- and Ets-1-binding sites in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-Sp1 or anti-Ets-1 antibodies, and the whole cell lysates (WCL) were monitored for the expression of Sp1 or Ets-1. The relative intensity of bands was quantified using the ImageGouge software (Fuji). (F) CHO cells treated with or not with hd-siRNAs were harvested for chromatin immunoprecipitation (ChIP) assays. Chromatin suspensions were immunoprecipitated with the indicated antibodies and control IgG. The resulting precipitants were subjected to PCR to amplify the region as indicated in the upper panel. The amplified products were visualized by ethidium bromide staining after gel electrophoresis. Representative data of 30 cycles are shown.
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pone-0026466-g003: Involvement of Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) Knockdown of Sp1 and Ets-1 genes inhibits the siRNA-stimulated meri-1 promoter activity. A: The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus siSp1_1, siSp1_2, siEts-1_1 or siEts-1_2 was measured. The knockdown effects were shown in lower panel. (B) Overexpression of the Sp1 and Ets-1 dominant-negative mutant inhibits the siRNA-stimulated meri-1 promoter activity. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus pCMV-HA, pCMV-HA-Sp1(DN) or pCMV-HA-Ets-1(DN) was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C)–(F) Sp1 and Ets-1 binding specifically to the meri-1 promoter. (C) and (D) EMSAs were performed using various biotin-labeled oligonucleotides corresponding to the putative Sp1 and Ets-1 elements in the meri-1 promoter, as described in Table 1. Nuclear extracts were prepared from CHO cells pre-treated with Nc siRNA_1. The competition assays were performed with or without a 50-fold excess of each unlabelled oligonucleotide, as indicated above each lane. Immunodepletion EMSAs were performed using 1 µl of the anti-Sp1, anti-Ets-1 antibodies and control normal mouse IgG, as indicated above each lane. Immunodepletion effects were shown under the related lanes. (E) Cell extracts of the CHO cells pre-treated with or not with Nc siRNA were prepared. The aliquots were subjected to DNA affinity precipitation assay (DAPA) with biotinylated oligonucleotides that contained the Sp1-, Ets-1- or both Sp1- and Ets-1-binding sites in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-Sp1 or anti-Ets-1 antibodies, and the whole cell lysates (WCL) were monitored for the expression of Sp1 or Ets-1. The relative intensity of bands was quantified using the ImageGouge software (Fuji). (F) CHO cells treated with or not with hd-siRNAs were harvested for chromatin immunoprecipitation (ChIP) assays. Chromatin suspensions were immunoprecipitated with the indicated antibodies and control IgG. The resulting precipitants were subjected to PCR to amplify the region as indicated in the upper panel. The amplified products were visualized by ethidium bromide staining after gel electrophoresis. Representative data of 30 cycles are shown.

Mentions: To examine the roles of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter activity, specific siRNAs were prepared to knock down Sp1 and Ets-1. These siRNAs were used to transfect CHO cells together with hd-siRNAs and pPeri-1(−87)-SEAP reporter plasmid. SEAP assays revealed that the knockdown of Sp1 and Ets-1 significantly decreased meri-1 promoter activity (Figure 3A). These results suggest that Sp1 and Ets-1 are involved in the regulation of hd-siRNA-stimulated meri-1 promoter activity.


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)

Involvement of Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) Knockdown of Sp1 and Ets-1 genes inhibits the siRNA-stimulated meri-1 promoter activity. A: The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus siSp1_1, siSp1_2, siEts-1_1 or siEts-1_2 was measured. The knockdown effects were shown in lower panel. (B) Overexpression of the Sp1 and Ets-1 dominant-negative mutant inhibits the siRNA-stimulated meri-1 promoter activity. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus pCMV-HA, pCMV-HA-Sp1(DN) or pCMV-HA-Ets-1(DN) was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C)–(F) Sp1 and Ets-1 binding specifically to the meri-1 promoter. (C) and (D) EMSAs were performed using various biotin-labeled oligonucleotides corresponding to the putative Sp1 and Ets-1 elements in the meri-1 promoter, as described in Table 1. Nuclear extracts were prepared from CHO cells pre-treated with Nc siRNA_1. The competition assays were performed with or without a 50-fold excess of each unlabelled oligonucleotide, as indicated above each lane. Immunodepletion EMSAs were performed using 1 µl of the anti-Sp1, anti-Ets-1 antibodies and control normal mouse IgG, as indicated above each lane. Immunodepletion effects were shown under the related lanes. (E) Cell extracts of the CHO cells pre-treated with or not with Nc siRNA were prepared. The aliquots were subjected to DNA affinity precipitation assay (DAPA) with biotinylated oligonucleotides that contained the Sp1-, Ets-1- or both Sp1- and Ets-1-binding sites in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-Sp1 or anti-Ets-1 antibodies, and the whole cell lysates (WCL) were monitored for the expression of Sp1 or Ets-1. The relative intensity of bands was quantified using the ImageGouge software (Fuji). (F) CHO cells treated with or not with hd-siRNAs were harvested for chromatin immunoprecipitation (ChIP) assays. Chromatin suspensions were immunoprecipitated with the indicated antibodies and control IgG. The resulting precipitants were subjected to PCR to amplify the region as indicated in the upper panel. The amplified products were visualized by ethidium bromide staining after gel electrophoresis. Representative data of 30 cycles are shown.
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pone-0026466-g003: Involvement of Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) Knockdown of Sp1 and Ets-1 genes inhibits the siRNA-stimulated meri-1 promoter activity. A: The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus siSp1_1, siSp1_2, siEts-1_1 or siEts-1_2 was measured. The knockdown effects were shown in lower panel. (B) Overexpression of the Sp1 and Ets-1 dominant-negative mutant inhibits the siRNA-stimulated meri-1 promoter activity. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA plus pCMV-HA, pCMV-HA-Sp1(DN) or pCMV-HA-Ets-1(DN) was measured. The data represents the mean of three independent experiments, and the error bars indicate the SD of triplicate samples. (C)–(F) Sp1 and Ets-1 binding specifically to the meri-1 promoter. (C) and (D) EMSAs were performed using various biotin-labeled oligonucleotides corresponding to the putative Sp1 and Ets-1 elements in the meri-1 promoter, as described in Table 1. Nuclear extracts were prepared from CHO cells pre-treated with Nc siRNA_1. The competition assays were performed with or without a 50-fold excess of each unlabelled oligonucleotide, as indicated above each lane. Immunodepletion EMSAs were performed using 1 µl of the anti-Sp1, anti-Ets-1 antibodies and control normal mouse IgG, as indicated above each lane. Immunodepletion effects were shown under the related lanes. (E) Cell extracts of the CHO cells pre-treated with or not with Nc siRNA were prepared. The aliquots were subjected to DNA affinity precipitation assay (DAPA) with biotinylated oligonucleotides that contained the Sp1-, Ets-1- or both Sp1- and Ets-1-binding sites in the meri-1 promoter (see Table 1). The precipitated complexes (Precipitation) were immunoblotted with anti-Sp1 or anti-Ets-1 antibodies, and the whole cell lysates (WCL) were monitored for the expression of Sp1 or Ets-1. The relative intensity of bands was quantified using the ImageGouge software (Fuji). (F) CHO cells treated with or not with hd-siRNAs were harvested for chromatin immunoprecipitation (ChIP) assays. Chromatin suspensions were immunoprecipitated with the indicated antibodies and control IgG. The resulting precipitants were subjected to PCR to amplify the region as indicated in the upper panel. The amplified products were visualized by ethidium bromide staining after gel electrophoresis. Representative data of 30 cycles are shown.
Mentions: To examine the roles of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter activity, specific siRNAs were prepared to knock down Sp1 and Ets-1. These siRNAs were used to transfect CHO cells together with hd-siRNAs and pPeri-1(−87)-SEAP reporter plasmid. SEAP assays revealed that the knockdown of Sp1 and Ets-1 significantly decreased meri-1 promoter activity (Figure 3A). These results suggest that Sp1 and Ets-1 are involved in the regulation of hd-siRNA-stimulated meri-1 promoter activity.

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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