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Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.

Haraguchi T, Ozaki Y, Iba H - Nucleic Acids Res. (2009)

Bottom Line: These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III.We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs).We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

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Related in: MedlinePlus

Generality, specificity and duration of the inhibitory effects of TuD RNA. (A) Effects of TuD-miR140-5p-4ntin or TuD-miR140-3p-4ntin on miR-140-3p activity detected by the GFP reporter cell system as shown in Table 1, except that the GFP expression levels were determined at 8–12 days post-transduction (Supplementary Figure S3D and E). (B) Effects of TuD-miR140-5p-4ntin or TuD-miR-140-3p-4ntin on miR-140-5p activity. HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector were transduced with the lentivirus vectors expressing the corresponding TuD RNAs and the GFP expression levels were determined at 8–12 days post-transduction. The expression levels were normalized to those of HeLaS3 cells harbouring miR-140-5p reporter alone and are represented by the mean ± SEM (n = 3). (C) Time course of the inhibitory effects of TuD-miR140-3p-4ntin on miR-140-3p activity. Relative GFP expression levels were determined as shown in Table 1. (D) Levels of mature miR-140-5p in the cells described in (B) determined by quantitative real-time RT-PCR. The miR-140-5p expression levels were normalized to those of HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) Analysis of the sub-cellular localization of TuD RNAs. The migration positions of Y4 small cytoplasmic RNA (Y4 scRNA, 93 nt) and ACA1 small nucleolar RNA (ACA1 snoRNA, 130 nt) on the same gel are indicated by black and open triangles, respectively. Y4 scRNA and ACA1 snoRNA were served as marker RNAs of cytoplasmic and nuclear fractions, respectively. Un, untransduced cells; 5p, TuD-miR140-5p-transduced cells; 3p, TuD-miR140-3p-transduced cells; N, nucleus; C, cytoplasm.
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Figure 3: Generality, specificity and duration of the inhibitory effects of TuD RNA. (A) Effects of TuD-miR140-5p-4ntin or TuD-miR140-3p-4ntin on miR-140-3p activity detected by the GFP reporter cell system as shown in Table 1, except that the GFP expression levels were determined at 8–12 days post-transduction (Supplementary Figure S3D and E). (B) Effects of TuD-miR140-5p-4ntin or TuD-miR-140-3p-4ntin on miR-140-5p activity. HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector were transduced with the lentivirus vectors expressing the corresponding TuD RNAs and the GFP expression levels were determined at 8–12 days post-transduction. The expression levels were normalized to those of HeLaS3 cells harbouring miR-140-5p reporter alone and are represented by the mean ± SEM (n = 3). (C) Time course of the inhibitory effects of TuD-miR140-3p-4ntin on miR-140-3p activity. Relative GFP expression levels were determined as shown in Table 1. (D) Levels of mature miR-140-5p in the cells described in (B) determined by quantitative real-time RT-PCR. The miR-140-5p expression levels were normalized to those of HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) Analysis of the sub-cellular localization of TuD RNAs. The migration positions of Y4 small cytoplasmic RNA (Y4 scRNA, 93 nt) and ACA1 small nucleolar RNA (ACA1 snoRNA, 130 nt) on the same gel are indicated by black and open triangles, respectively. Y4 scRNA and ACA1 snoRNA were served as marker RNAs of cytoplasmic and nuclear fractions, respectively. Un, untransduced cells; 5p, TuD-miR140-5p-transduced cells; 3p, TuD-miR140-3p-transduced cells; N, nucleus; C, cytoplasm.

Mentions: To test the generality and specificity of the inhibitory effects of TuD RNA, we constructed a reporter cell system for miR-140-5p in which the insertion sequence of the miR-140-3p reporter was substituted with a sequence that was fully complementary to miR-140-5p (Supplementary Figure S1B). The inhibitory effects of TuD-miR140-3p, harbouring an MBS with four extra nucleotides (TuD-miR140-3p-4ntin; Decoy RNA#020) and TuD-miR140-5p-4ntin (Supplementary Figure S4) were determined using both the miR-140-3p and miR-140-5p reporter cell systems. In the miR-140-3p reporter system, TuD-miR140-3p-4ntin introduction resulted in a complete recovery of GFP expression but TuD-miR140-5p-4ntin expression showed only marginal effects (Figure 3A and Supplementary Figure S2D and E). In the miR-140-5p reporter system, the converse result was obtained with these two TuD RNA constructs (Figure 3B). These results indicate that the inhibitory effects of these TuD RNAs are highly efficient and specific to their target miRNAs, lending further support to the wide applicability of these molecules to miRNA suppression.Figure 3.


Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells.

Haraguchi T, Ozaki Y, Iba H - Nucleic Acids Res. (2009)

Generality, specificity and duration of the inhibitory effects of TuD RNA. (A) Effects of TuD-miR140-5p-4ntin or TuD-miR140-3p-4ntin on miR-140-3p activity detected by the GFP reporter cell system as shown in Table 1, except that the GFP expression levels were determined at 8–12 days post-transduction (Supplementary Figure S3D and E). (B) Effects of TuD-miR140-5p-4ntin or TuD-miR-140-3p-4ntin on miR-140-5p activity. HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector were transduced with the lentivirus vectors expressing the corresponding TuD RNAs and the GFP expression levels were determined at 8–12 days post-transduction. The expression levels were normalized to those of HeLaS3 cells harbouring miR-140-5p reporter alone and are represented by the mean ± SEM (n = 3). (C) Time course of the inhibitory effects of TuD-miR140-3p-4ntin on miR-140-3p activity. Relative GFP expression levels were determined as shown in Table 1. (D) Levels of mature miR-140-5p in the cells described in (B) determined by quantitative real-time RT-PCR. The miR-140-5p expression levels were normalized to those of HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) Analysis of the sub-cellular localization of TuD RNAs. The migration positions of Y4 small cytoplasmic RNA (Y4 scRNA, 93 nt) and ACA1 small nucleolar RNA (ACA1 snoRNA, 130 nt) on the same gel are indicated by black and open triangles, respectively. Y4 scRNA and ACA1 snoRNA were served as marker RNAs of cytoplasmic and nuclear fractions, respectively. Un, untransduced cells; 5p, TuD-miR140-5p-transduced cells; 3p, TuD-miR140-3p-transduced cells; N, nucleus; C, cytoplasm.
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Figure 3: Generality, specificity and duration of the inhibitory effects of TuD RNA. (A) Effects of TuD-miR140-5p-4ntin or TuD-miR140-3p-4ntin on miR-140-3p activity detected by the GFP reporter cell system as shown in Table 1, except that the GFP expression levels were determined at 8–12 days post-transduction (Supplementary Figure S3D and E). (B) Effects of TuD-miR140-5p-4ntin or TuD-miR-140-3p-4ntin on miR-140-5p activity. HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector were transduced with the lentivirus vectors expressing the corresponding TuD RNAs and the GFP expression levels were determined at 8–12 days post-transduction. The expression levels were normalized to those of HeLaS3 cells harbouring miR-140-5p reporter alone and are represented by the mean ± SEM (n = 3). (C) Time course of the inhibitory effects of TuD-miR140-3p-4ntin on miR-140-3p activity. Relative GFP expression levels were determined as shown in Table 1. (D) Levels of mature miR-140-5p in the cells described in (B) determined by quantitative real-time RT-PCR. The miR-140-5p expression levels were normalized to those of HeLaS3 cells harbouring both the miR-140-5p reporter and the miR140-5p/140-3p vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) Analysis of the sub-cellular localization of TuD RNAs. The migration positions of Y4 small cytoplasmic RNA (Y4 scRNA, 93 nt) and ACA1 small nucleolar RNA (ACA1 snoRNA, 130 nt) on the same gel are indicated by black and open triangles, respectively. Y4 scRNA and ACA1 snoRNA were served as marker RNAs of cytoplasmic and nuclear fractions, respectively. Un, untransduced cells; 5p, TuD-miR140-5p-transduced cells; 3p, TuD-miR140-3p-transduced cells; N, nucleus; C, cytoplasm.
Mentions: To test the generality and specificity of the inhibitory effects of TuD RNA, we constructed a reporter cell system for miR-140-5p in which the insertion sequence of the miR-140-3p reporter was substituted with a sequence that was fully complementary to miR-140-5p (Supplementary Figure S1B). The inhibitory effects of TuD-miR140-3p, harbouring an MBS with four extra nucleotides (TuD-miR140-3p-4ntin; Decoy RNA#020) and TuD-miR140-5p-4ntin (Supplementary Figure S4) were determined using both the miR-140-3p and miR-140-5p reporter cell systems. In the miR-140-3p reporter system, TuD-miR140-3p-4ntin introduction resulted in a complete recovery of GFP expression but TuD-miR140-5p-4ntin expression showed only marginal effects (Figure 3A and Supplementary Figure S2D and E). In the miR-140-5p reporter system, the converse result was obtained with these two TuD RNA constructs (Figure 3B). These results indicate that the inhibitory effects of these TuD RNAs are highly efficient and specific to their target miRNAs, lending further support to the wide applicability of these molecules to miRNA suppression.Figure 3.

Bottom Line: These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III.We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs).We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT
Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells.

Show MeSH
Related in: MedlinePlus