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Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs.

Lebbink RJ, Lowe M, Chan T, Khine H, Wang X, McManus MT - PLoS ONE (2011)

Bottom Line: One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs).Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells.Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

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Relative quantity of anti-EGFP siRNAs correlates with target-knockdown.EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoters. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Total RNA was isolated and relative mature anti-EGFP siRNA expression was determined by TaqMan analysis. The lowest quantity of mature anti-EGFP within each cell line was set to 1. Relative mature anti-EGFP siRNA expression is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.
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pone-0026213-g006: Relative quantity of anti-EGFP siRNAs correlates with target-knockdown.EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoters. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Total RNA was isolated and relative mature anti-EGFP siRNA expression was determined by TaqMan analysis. The lowest quantity of mature anti-EGFP within each cell line was set to 1. Relative mature anti-EGFP siRNA expression is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.

Mentions: If promoter strength is a critical variable for induction of potent RNAi, the amount of small RNAs should correlate with the activity of the promoter. To formally test this we quantitated the relative expression levels of the fully processed anti-EGFP siRNA antisense strand in the cells expressing the various promoter constructs. To this end, we designed TaqMan qPCR primers and probes specific for the ‘mature’ anti-EGFP siRNA based on the validated TaqMan approach for mature miRNAs [28]. The qPCR was specific for the mature anti-EGFP siRNA strand (data not shown) and efficiently amplified this species in our cell lines expressing anti-EGFP shRNAs driven by either the U6 promoter or the various polymerase II promoters. We sorted cells expressing single copies of the various anti-EGFP expressing shRNAs vectors by FACS, and assessed the relative expression level of mature anti-EGFP siRNAs herein and plotted this to the percentage of protein target knockdown at this same timepoint (see Figure 6). Again, for all lines, there was a clear correlation between promoter strength (as determined by mature anti-EGFP siRNA expression) and EGFP-knockdown by the anti-EGFP shRNA. We conclude that a more potent polymerase II promoter yields higher quantities of anti-target siRNAs, resulting in higher protein target knockdown. Of note, since we quantitated the EGFP target knockdown by measuring the amount of fluorescent protein in the cells and not by assessing the EGFP mRNA levels, we cannot account for the impact of differences in EGFP stability on RNAi in these different cell types.


Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs.

Lebbink RJ, Lowe M, Chan T, Khine H, Wang X, McManus MT - PLoS ONE (2011)

Relative quantity of anti-EGFP siRNAs correlates with target-knockdown.EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoters. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Total RNA was isolated and relative mature anti-EGFP siRNA expression was determined by TaqMan analysis. The lowest quantity of mature anti-EGFP within each cell line was set to 1. Relative mature anti-EGFP siRNA expression is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198731&req=5

pone-0026213-g006: Relative quantity of anti-EGFP siRNAs correlates with target-knockdown.EGFP-expressing human immune cell types (Raji B, Jurkat T, and THP-1 monocytic cells) and adherent cell lines (293T, HeLa, and HT29 cells) were infected at an MOI of <0.2 with anti-EGFP shRNAs (left panels) expressed from a miR-30 backbone driven by various polymerase-II promoters. Cells were allowed to grow for 8 days and the expression of EGFP (to monitor knockdown) was assessed by flow cytometry. Total RNA was isolated and relative mature anti-EGFP siRNA expression was determined by TaqMan analysis. The lowest quantity of mature anti-EGFP within each cell line was set to 1. Relative mature anti-EGFP siRNA expression is plotted against% EGFP knockdown in the indicated cellines. Trendlines and R2 values of fitted curves are indicated.
Mentions: If promoter strength is a critical variable for induction of potent RNAi, the amount of small RNAs should correlate with the activity of the promoter. To formally test this we quantitated the relative expression levels of the fully processed anti-EGFP siRNA antisense strand in the cells expressing the various promoter constructs. To this end, we designed TaqMan qPCR primers and probes specific for the ‘mature’ anti-EGFP siRNA based on the validated TaqMan approach for mature miRNAs [28]. The qPCR was specific for the mature anti-EGFP siRNA strand (data not shown) and efficiently amplified this species in our cell lines expressing anti-EGFP shRNAs driven by either the U6 promoter or the various polymerase II promoters. We sorted cells expressing single copies of the various anti-EGFP expressing shRNAs vectors by FACS, and assessed the relative expression level of mature anti-EGFP siRNAs herein and plotted this to the percentage of protein target knockdown at this same timepoint (see Figure 6). Again, for all lines, there was a clear correlation between promoter strength (as determined by mature anti-EGFP siRNA expression) and EGFP-knockdown by the anti-EGFP shRNA. We conclude that a more potent polymerase II promoter yields higher quantities of anti-target siRNAs, resulting in higher protein target knockdown. Of note, since we quantitated the EGFP target knockdown by measuring the amount of fluorescent protein in the cells and not by assessing the EGFP mRNA levels, we cannot account for the impact of differences in EGFP stability on RNAi in these different cell types.

Bottom Line: One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs).Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells.Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.

Show MeSH