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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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STAT3 bridges Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) The siRNA-stimulated meri-1 promoter activity is further enhanced by overexpression of STAT3. By using various STAT3 mutants, the involvement of STAT3 was examined. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus the indicating plasmids expressing Sp1, Ets-1, STAT3 and various STAT3 mutants. (B) Knockdown of STAT3 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 siSTAT3_1 or siSTAT3_2 was measured. (C) STAT3 is recruited to the meri-1 promoter by hd-siRNA induction. Cell extracts of the CHO cells transfected with or without hd-siRNA were prepared. The aliquots were subjected to 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-STAT3 antibody, and the whole cell lysates (WCL) were monitored for the expression of STAT3. The relative intensity of bands was quantified using the ImageGouge software. (D) STAT3 functions in the meri-1 promoter activity without DNA binding. Cell extracts of the CHO cells transfected with Nc siRNA_2, or with Nc siRNA_2 plus siSp1_2 and siEts-1_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in meri-1 promoter (see Table 1). (E) STAT3 functions as a bridge of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter activity. Cell extracts were prepared from the CHO cells co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2 plus siSTAT3_2 or not. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-Ets-1 antibody. The relative intensity of bands was quantified using the ImageGouge software.
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pone-0026466-g005: STAT3 bridges Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) The siRNA-stimulated meri-1 promoter activity is further enhanced by overexpression of STAT3. By using various STAT3 mutants, the involvement of STAT3 was examined. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus the indicating plasmids expressing Sp1, Ets-1, STAT3 and various STAT3 mutants. (B) Knockdown of STAT3 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 siSTAT3_1 or siSTAT3_2 was measured. (C) STAT3 is recruited to the meri-1 promoter by hd-siRNA induction. Cell extracts of the CHO cells transfected with or without hd-siRNA were prepared. The aliquots were subjected to 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-STAT3 antibody, and the whole cell lysates (WCL) were monitored for the expression of STAT3. The relative intensity of bands was quantified using the ImageGouge software. (D) STAT3 functions in the meri-1 promoter activity without DNA binding. Cell extracts of the CHO cells transfected with Nc siRNA_2, or with Nc siRNA_2 plus siSp1_2 and siEts-1_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in meri-1 promoter (see Table 1). (E) STAT3 functions as a bridge of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter activity. Cell extracts were prepared from the CHO cells co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2 plus siSTAT3_2 or not. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-Ets-1 antibody. The relative intensity of bands was quantified using the ImageGouge software.

Mentions: Because overexpression of Sp1, Ets-1 or Sp1 and Ets-1 could only increase meri-1 transcription very slightly (Figure 5A: panels 2–4) , the later studies, which have shown that siRNAs can activate innate immunity in mammalian cells [57], [58], [59], [60], [61], prompted us to examine the involvement of inflammation-related transcription factors in the regulation of the hd-siRNA-stimulated meri-1expression. Through screening by RNAi, we found that the STAT3 knockdown significantly decreased the meri-1 promoter activity (Figure 5B: panel 2 and 3), while the overexpression of STAT3 significantly increased the meri-1 promoter activity (Figure 5A: panel 5). Co-expression of Sp1, Ets-1 and STAT3 increased the meri-1 promoter activity to a higher level than Sp1 and/or Ets-1 alone (Figure 5A: panel 6), suggesting that STAT3 is essential for the 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)

STAT3 bridges Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) The siRNA-stimulated meri-1 promoter activity is further enhanced by overexpression of STAT3. By using various STAT3 mutants, the involvement of STAT3 was examined. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus the indicating plasmids expressing Sp1, Ets-1, STAT3 and various STAT3 mutants. (B) Knockdown of STAT3 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 siSTAT3_1 or siSTAT3_2 was measured. (C) STAT3 is recruited to the meri-1 promoter by hd-siRNA induction. Cell extracts of the CHO cells transfected with or without hd-siRNA were prepared. The aliquots were subjected to 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-STAT3 antibody, and the whole cell lysates (WCL) were monitored for the expression of STAT3. The relative intensity of bands was quantified using the ImageGouge software. (D) STAT3 functions in the meri-1 promoter activity without DNA binding. Cell extracts of the CHO cells transfected with Nc siRNA_2, or with Nc siRNA_2 plus siSp1_2 and siEts-1_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in meri-1 promoter (see Table 1). (E) STAT3 functions as a bridge of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter activity. Cell extracts were prepared from the CHO cells co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2 plus siSTAT3_2 or not. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-Ets-1 antibody. The relative intensity of bands was quantified using the ImageGouge software.
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pone-0026466-g005: STAT3 bridges Sp1 and Ets-1 in the siRNA-stimulated meri-1 Promoter Activity.(A) The siRNA-stimulated meri-1 promoter activity is further enhanced by overexpression of STAT3. By using various STAT3 mutants, the involvement of STAT3 was examined. The SEAP activity in the culture media of the CHO cells co-transfected with pPeri-1(−87)-SEAP and Nc siRNA_2 plus the indicating plasmids expressing Sp1, Ets-1, STAT3 and various STAT3 mutants. (B) Knockdown of STAT3 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 siSTAT3_1 or siSTAT3_2 was measured. (C) STAT3 is recruited to the meri-1 promoter by hd-siRNA induction. Cell extracts of the CHO cells transfected with or without hd-siRNA were prepared. The aliquots were subjected to 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-STAT3 antibody, and the whole cell lysates (WCL) were monitored for the expression of STAT3. The relative intensity of bands was quantified using the ImageGouge software. (D) STAT3 functions in the meri-1 promoter activity without DNA binding. Cell extracts of the CHO cells transfected with Nc siRNA_2, or with Nc siRNA_2 plus siSp1_2 and siEts-1_2 were prepared. The aliquots were subjected to DAPA with biotinylated Peri-1(−88–−30) oligonucleotides that contain the hd-siRNA elements in meri-1 promoter (see Table 1). (E) STAT3 functions as a bridge of Sp1 and Ets-1 in the hd-siRNA-stimulated meri-1 promoter activity. Cell extracts were prepared from the CHO cells co-transfected with pCMV-FLAG-Sp1 and Nc siRNA_2 plus siSTAT3_2 or not. Immunoprecipitation was performed using M2-anti-FLAG antibody-agarose beads. The immunoprecipitated pellets were analyzed by immunoblotting with anti-Ets-1 antibody. The relative intensity of bands was quantified using the ImageGouge software.
Mentions: Because overexpression of Sp1, Ets-1 or Sp1 and Ets-1 could only increase meri-1 transcription very slightly (Figure 5A: panels 2–4) , the later studies, which have shown that siRNAs can activate innate immunity in mammalian cells [57], [58], [59], [60], [61], prompted us to examine the involvement of inflammation-related transcription factors in the regulation of the hd-siRNA-stimulated meri-1expression. Through screening by RNAi, we found that the STAT3 knockdown significantly decreased the meri-1 promoter activity (Figure 5B: panel 2 and 3), while the overexpression of STAT3 significantly increased the meri-1 promoter activity (Figure 5A: panel 5). Co-expression of Sp1, Ets-1 and STAT3 increased the meri-1 promoter activity to a higher level than Sp1 and/or Ets-1 alone (Figure 5A: panel 6), suggesting that STAT3 is essential for the 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.

Show MeSH