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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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Characterization of the meri-1 promoter Sequence.(A) Upregulation of meri-1 transcription induced by hd-siRNAs in CHO cells. CHO cells were transfected with indicated quantity of Nc siRNA_2. The meri-1 mRNA level was measured by qPCR, twelve hours after transfection. (B) Time course of hd-siRNA-stimulated transcription of meri-1. CHO cells were transfected with Nc siRNA_2. The meri-1 mRNA level was measured by qPCR. (C) Determination of the responsive ability of the 1.8 kb meri-1 promoter, Peri-1(−1654), to hd-siRNAs. CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (siHBVP) (II), or 21-bp DNA control (I). (D) Response of the meri-1 promoter to enzymatically synthesized siRNA (Nc siRNA_1) and chemically synthesized siRNA (Nc siRNA_2). CHO cells were co-transfected with SEAP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (panel 1), Nc siRNA_2 (panel 2), or the 21-bp DNA control (panel 3). (E) Response of the meri-1 promoter to hd-siRNA in the HEK 293 cells. (F) The necessity of liposome-mediated transfection process for the hd-siRNA-stimulated meri-1 promoter activity. CHO cells were transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter. Four hours later, the medium was removed and the cells were washed three times by fresh medium. Then, Nc siRNA_1 was added to the medium without transfection agent (I). CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 was used as a control (II).
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pone-0026466-g001: Characterization of the meri-1 promoter Sequence.(A) Upregulation of meri-1 transcription induced by hd-siRNAs in CHO cells. CHO cells were transfected with indicated quantity of Nc siRNA_2. The meri-1 mRNA level was measured by qPCR, twelve hours after transfection. (B) Time course of hd-siRNA-stimulated transcription of meri-1. CHO cells were transfected with Nc siRNA_2. The meri-1 mRNA level was measured by qPCR. (C) Determination of the responsive ability of the 1.8 kb meri-1 promoter, Peri-1(−1654), to hd-siRNAs. CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (siHBVP) (II), or 21-bp DNA control (I). (D) Response of the meri-1 promoter to enzymatically synthesized siRNA (Nc siRNA_1) and chemically synthesized siRNA (Nc siRNA_2). CHO cells were co-transfected with SEAP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (panel 1), Nc siRNA_2 (panel 2), or the 21-bp DNA control (panel 3). (E) Response of the meri-1 promoter to hd-siRNA in the HEK 293 cells. (F) The necessity of liposome-mediated transfection process for the hd-siRNA-stimulated meri-1 promoter activity. CHO cells were transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter. Four hours later, the medium was removed and the cells were washed three times by fresh medium. Then, Nc siRNA_1 was added to the medium without transfection agent (I). CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 was used as a control (II).

Mentions: Our previous study has showed that I.V. injection of hd-siRNAs upregulates the meri-1 transcription in mouse liver [10]. To analyze the in vitro meri-1 transcription in response to hd-siRNA treatment, we measured meri-1 mRNA level by qPCR in CHO cells. As shown in Figure 1A, meri-1 transcription was significantly upregulated in CHO cells treated with >0.4 µg/24-well plate of Nc siRNA. This meri-1 transcription peaked at 12 h after Nc siRNA transfection and gradually decreased to its basal level after 36 h (Figure 1B). Therefore, the following experiments were carried out using 1 µg/24-well plate of Nc siRNA and data was collected 24 h after Nc siRNA transfection.


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)

Characterization of the meri-1 promoter Sequence.(A) Upregulation of meri-1 transcription induced by hd-siRNAs in CHO cells. CHO cells were transfected with indicated quantity of Nc siRNA_2. The meri-1 mRNA level was measured by qPCR, twelve hours after transfection. (B) Time course of hd-siRNA-stimulated transcription of meri-1. CHO cells were transfected with Nc siRNA_2. The meri-1 mRNA level was measured by qPCR. (C) Determination of the responsive ability of the 1.8 kb meri-1 promoter, Peri-1(−1654), to hd-siRNAs. CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (siHBVP) (II), or 21-bp DNA control (I). (D) Response of the meri-1 promoter to enzymatically synthesized siRNA (Nc siRNA_1) and chemically synthesized siRNA (Nc siRNA_2). CHO cells were co-transfected with SEAP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (panel 1), Nc siRNA_2 (panel 2), or the 21-bp DNA control (panel 3). (E) Response of the meri-1 promoter to hd-siRNA in the HEK 293 cells. (F) The necessity of liposome-mediated transfection process for the hd-siRNA-stimulated meri-1 promoter activity. CHO cells were transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter. Four hours later, the medium was removed and the cells were washed three times by fresh medium. Then, Nc siRNA_1 was added to the medium without transfection agent (I). CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 was used as a control (II).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198429&req=5

pone-0026466-g001: Characterization of the meri-1 promoter Sequence.(A) Upregulation of meri-1 transcription induced by hd-siRNAs in CHO cells. CHO cells were transfected with indicated quantity of Nc siRNA_2. The meri-1 mRNA level was measured by qPCR, twelve hours after transfection. (B) Time course of hd-siRNA-stimulated transcription of meri-1. CHO cells were transfected with Nc siRNA_2. The meri-1 mRNA level was measured by qPCR. (C) Determination of the responsive ability of the 1.8 kb meri-1 promoter, Peri-1(−1654), to hd-siRNAs. CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (siHBVP) (II), or 21-bp DNA control (I). (D) Response of the meri-1 promoter to enzymatically synthesized siRNA (Nc siRNA_1) and chemically synthesized siRNA (Nc siRNA_2). CHO cells were co-transfected with SEAP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 (panel 1), Nc siRNA_2 (panel 2), or the 21-bp DNA control (panel 3). (E) Response of the meri-1 promoter to hd-siRNA in the HEK 293 cells. (F) The necessity of liposome-mediated transfection process for the hd-siRNA-stimulated meri-1 promoter activity. CHO cells were transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter. Four hours later, the medium was removed and the cells were washed three times by fresh medium. Then, Nc siRNA_1 was added to the medium without transfection agent (I). CHO cells were co-transfected with GFP reporter construct containing the 1.8 kb meri-1 promoter and Nc siRNA_1 was used as a control (II).
Mentions: Our previous study has showed that I.V. injection of hd-siRNAs upregulates the meri-1 transcription in mouse liver [10]. To analyze the in vitro meri-1 transcription in response to hd-siRNA treatment, we measured meri-1 mRNA level by qPCR in CHO cells. As shown in Figure 1A, meri-1 transcription was significantly upregulated in CHO cells treated with >0.4 µg/24-well plate of Nc siRNA. This meri-1 transcription peaked at 12 h after Nc siRNA transfection and gradually decreased to its basal level after 36 h (Figure 1B). Therefore, the following experiments were carried out using 1 µg/24-well plate of Nc siRNA and data was collected 24 h after Nc siRNA transfection.

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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