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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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3′-UTR involved in the upregulation of meri-1 expression stimulated by hd-siRNA.(A) 3′-UTR of meri-1 functions in the regulation of meri-1 expression. The SEAP activity in the culture media of the CHO cells transfected with SEAP constitutive expression construct, pSEAP2-control, or with SEAP reporter construct containing the 3.9 kb meri-1 3′-UTR was measured. (B) Quantity of SEAP mRNA unaffected by meri-1 3′-UTR. The SEAP mRNA level was measured by qPCR. (C) Nc siRNAs reduced the meri-1 3′-UTR-mediated repression in a dosage-dependent manner. The SEAP activity in the culture media of the CHO cells co-transfected with pSEAP-meri-1_3′-UTR and an increasing dose of Nc siRNA_2 was measured. (D) Quantity of SEAP-meri-1_3′-UTR mRNA unaffected by hd-siRNA treatment. The SEAP-meri-1_3′-UTR mRNA level was measured by qPCR. (E) Abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells that were transfected with an increasing dose of pSEAP-meri-1_3′-UTR plus Nc siRNA_2 (line2) or not plus Nc siRNA_2 (line1) was measured. (F) Time course of the abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells transfected with pSEAP-meri-1_3′-UTR plus Nc siRNA_2 or not plus Nc siRNA_2 was measured at the indicated hours after transfection.
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pone-0026466-g006: 3′-UTR involved in the upregulation of meri-1 expression stimulated by hd-siRNA.(A) 3′-UTR of meri-1 functions in the regulation of meri-1 expression. The SEAP activity in the culture media of the CHO cells transfected with SEAP constitutive expression construct, pSEAP2-control, or with SEAP reporter construct containing the 3.9 kb meri-1 3′-UTR was measured. (B) Quantity of SEAP mRNA unaffected by meri-1 3′-UTR. The SEAP mRNA level was measured by qPCR. (C) Nc siRNAs reduced the meri-1 3′-UTR-mediated repression in a dosage-dependent manner. The SEAP activity in the culture media of the CHO cells co-transfected with pSEAP-meri-1_3′-UTR and an increasing dose of Nc siRNA_2 was measured. (D) Quantity of SEAP-meri-1_3′-UTR mRNA unaffected by hd-siRNA treatment. The SEAP-meri-1_3′-UTR mRNA level was measured by qPCR. (E) Abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells that were transfected with an increasing dose of pSEAP-meri-1_3′-UTR plus Nc siRNA_2 (line2) or not plus Nc siRNA_2 (line1) was measured. (F) Time course of the abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells transfected with pSEAP-meri-1_3′-UTR plus Nc siRNA_2 or not plus Nc siRNA_2 was measured at the indicated hours after transfection.

Mentions: To examine whether the 3′-UTR participates in the regulation of meri-1 expression, equal molar amounts of the reporter plasmid, pSEAP-meri-1_3′-UTR (0.3 µg/24-well plate), and control plasmid, pSEAP2-control (0.17 µg/24-well plate), were transfected into CHO cells. As shown in Figure 6A, the expression of SEAP was significantly repressed by the 3′-UTR of meri-1. This indicates that the 3′-UTR of meri-1 mediates the gene repression. There were no differences in the quantity of SEAP mRNA from constructs with or without the meri-1 3′-UTR as assessed by qPCR (Figure 6 B). This indicates that the regulation is posttranscriptional and works in an expression repression manner rather than an mRNA destabilization manner. To determine whether hd-siRNAs affected this repression, CHO cells were cotransfected with 0.3 µg of pSEAP-meri-1_3′-UTR and increasing doses of Nc siRNA_2. Repression was reduced by Nc siRNA in a dosage-dependent manner until a limit at approximately 0.6 µg/24-well plate (Figure 6C). The mRNA level of SEAP-meri-1_3′-UTR was unchanged by hd-siRNA treatment, as assessed by qPCR assay (Figure 6D), suggesting that the effect of hd-siRNA occurs at the posttranscription level. To elucidate more details of this process, we transfected a linear increasing dose of pSEAP-meri-1_3′-UTR with or without 0.6 µg Nc siRNA. As shown in Figure 6E, the increase of SEAP expression was not proportional to the dose of reporter plasmid (Figure 6E: line 1). A threshold of the repression effect, at about 0.4 µg/24-well plate of pSEAP-meri-1_3′-UTR, appeared, below which the expression of SEAP was repressed. However, with hd-siRNA treatment (Figure 6E: line 2), the inflection point appeared earlier than compared with no siRNA treatment (Figure 6E: line 1). This result confirmed that hd-siRNA could abolish the repression of meri-1 expression exerted from its 3′-UTR. Time course analysis showed that the increase of reporter gene expression was in a time-dependent manner until it peaked at 12 h after Nc siRNA transfection (Figure 6F). Because the first four hours likely was the process of transfection, the results indicate that hd-siRNA exerted its effect rapidly. These results suggested that hd-siRNA could also abolish the posttranscription repression exerted from 3′-UTR, which further enhanced the expression of meri-1.


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)

3′-UTR involved in the upregulation of meri-1 expression stimulated by hd-siRNA.(A) 3′-UTR of meri-1 functions in the regulation of meri-1 expression. The SEAP activity in the culture media of the CHO cells transfected with SEAP constitutive expression construct, pSEAP2-control, or with SEAP reporter construct containing the 3.9 kb meri-1 3′-UTR was measured. (B) Quantity of SEAP mRNA unaffected by meri-1 3′-UTR. The SEAP mRNA level was measured by qPCR. (C) Nc siRNAs reduced the meri-1 3′-UTR-mediated repression in a dosage-dependent manner. The SEAP activity in the culture media of the CHO cells co-transfected with pSEAP-meri-1_3′-UTR and an increasing dose of Nc siRNA_2 was measured. (D) Quantity of SEAP-meri-1_3′-UTR mRNA unaffected by hd-siRNA treatment. The SEAP-meri-1_3′-UTR mRNA level was measured by qPCR. (E) Abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells that were transfected with an increasing dose of pSEAP-meri-1_3′-UTR plus Nc siRNA_2 (line2) or not plus Nc siRNA_2 (line1) was measured. (F) Time course of the abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells transfected with pSEAP-meri-1_3′-UTR plus Nc siRNA_2 or not plus Nc siRNA_2 was measured at the indicated hours after transfection.
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Related In: Results  -  Collection

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pone-0026466-g006: 3′-UTR involved in the upregulation of meri-1 expression stimulated by hd-siRNA.(A) 3′-UTR of meri-1 functions in the regulation of meri-1 expression. The SEAP activity in the culture media of the CHO cells transfected with SEAP constitutive expression construct, pSEAP2-control, or with SEAP reporter construct containing the 3.9 kb meri-1 3′-UTR was measured. (B) Quantity of SEAP mRNA unaffected by meri-1 3′-UTR. The SEAP mRNA level was measured by qPCR. (C) Nc siRNAs reduced the meri-1 3′-UTR-mediated repression in a dosage-dependent manner. The SEAP activity in the culture media of the CHO cells co-transfected with pSEAP-meri-1_3′-UTR and an increasing dose of Nc siRNA_2 was measured. (D) Quantity of SEAP-meri-1_3′-UTR mRNA unaffected by hd-siRNA treatment. The SEAP-meri-1_3′-UTR mRNA level was measured by qPCR. (E) Abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells that were transfected with an increasing dose of pSEAP-meri-1_3′-UTR plus Nc siRNA_2 (line2) or not plus Nc siRNA_2 (line1) was measured. (F) Time course of the abolishment of the meri-1 3′-UTR-mediated repression by hd-siRNA treatment. The SEAP activity in the culture media of the CHO cells transfected with pSEAP-meri-1_3′-UTR plus Nc siRNA_2 or not plus Nc siRNA_2 was measured at the indicated hours after transfection.
Mentions: To examine whether the 3′-UTR participates in the regulation of meri-1 expression, equal molar amounts of the reporter plasmid, pSEAP-meri-1_3′-UTR (0.3 µg/24-well plate), and control plasmid, pSEAP2-control (0.17 µg/24-well plate), were transfected into CHO cells. As shown in Figure 6A, the expression of SEAP was significantly repressed by the 3′-UTR of meri-1. This indicates that the 3′-UTR of meri-1 mediates the gene repression. There were no differences in the quantity of SEAP mRNA from constructs with or without the meri-1 3′-UTR as assessed by qPCR (Figure 6 B). This indicates that the regulation is posttranscriptional and works in an expression repression manner rather than an mRNA destabilization manner. To determine whether hd-siRNAs affected this repression, CHO cells were cotransfected with 0.3 µg of pSEAP-meri-1_3′-UTR and increasing doses of Nc siRNA_2. Repression was reduced by Nc siRNA in a dosage-dependent manner until a limit at approximately 0.6 µg/24-well plate (Figure 6C). The mRNA level of SEAP-meri-1_3′-UTR was unchanged by hd-siRNA treatment, as assessed by qPCR assay (Figure 6D), suggesting that the effect of hd-siRNA occurs at the posttranscription level. To elucidate more details of this process, we transfected a linear increasing dose of pSEAP-meri-1_3′-UTR with or without 0.6 µg Nc siRNA. As shown in Figure 6E, the increase of SEAP expression was not proportional to the dose of reporter plasmid (Figure 6E: line 1). A threshold of the repression effect, at about 0.4 µg/24-well plate of pSEAP-meri-1_3′-UTR, appeared, below which the expression of SEAP was repressed. However, with hd-siRNA treatment (Figure 6E: line 2), the inflection point appeared earlier than compared with no siRNA treatment (Figure 6E: line 1). This result confirmed that hd-siRNA could abolish the repression of meri-1 expression exerted from its 3′-UTR. Time course analysis showed that the increase of reporter gene expression was in a time-dependent manner until it peaked at 12 h after Nc siRNA transfection (Figure 6F). Because the first four hours likely was the process of transfection, the results indicate that hd-siRNA exerted its effect rapidly. These results suggested that hd-siRNA could also abolish the posttranscription repression exerted from 3′-UTR, which further enhanced the expression of meri-1.

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