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Atypical transcription of microRNA gene fragments.

Song Gao J, Zhang Y, Li M, Tucker LD, Machan JT, Quesenberry P, Rigoutsos I, Ramratnam B - Nucleic Acids Res. (2010)

Bottom Line: Here, we report that, in the absence of exogenous promoters, DNA fragments incorporating miRNA precursors can be delivered directly into a variety of human cells and give rise to the corresponding mature miRNA.Notably, the transcription of these miRNA DNA fragments appears resistant to conventional inhibitors of RNAP I/II/III activity.Taken together, our findings suggest the existence of a previously unrecognized atypical transcription program for miRNA precursor sequences.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Rhode Island and Miriam Hospitals, Warren Alpert Medical School, Brown University, Providence, RI 02903, USA.

ABSTRACT
MicroRNAs (miRNAs) are short ( approximately 22 nt) RNAs that impact gene expression by sequence-specific interactions with messenger RNA or promoter sequences of genomic DNA. Ectopic expression of miRNAs can be accomplished by placing fragments of the corresponding miRNA precursor under the control of RNA polymerase II or III (RNAP II/III). Here, we report that, in the absence of exogenous promoters, DNA fragments incorporating miRNA precursors can be delivered directly into a variety of human cells and give rise to the corresponding mature miRNA. Notably, the transcription of these miRNA DNA fragments appears resistant to conventional inhibitors of RNAP I/II/III activity. Taken together, our findings suggest the existence of a previously unrecognized atypical transcription program for miRNA precursor sequences.

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Related in: MedlinePlus

Functional activity of AmpmiRNA-XX. Luciferase-based miRNA sensor assays were used to compare the functional activity of various miRNA expression units. We created reporter constructs, in which the exact target sequence of a given miRNA was introduced into the 3′UTR of the gene encoding Renilla luciferase and quantified the relative reduction in luciferase levels compared to control experiments involving the same sensor but an empty vector (Ø). An internal firefly luciferase gene served to normalize data. All sensor assays were performed as three independent experiments and data are shown as mean reduction +/– SD compared to control conditions. (a) Functional activity of AmpmiRNA-143, AmpCMV-miRNA-143 and PlaCMV-miRNA-143. Co-transfection of the miRNA-143 sensor and AmpmiRNA-143 led to 57% decrease in luciferase activity, a statistically significant level (P = 0.02) approaching that achieved in transfections using a CMV-driven miRNA-143 expression plasmid (81% reduction) or amplicon (70% reduction) in HEK 293T cells. (b) Functional activity of shorter amplicons AmpmiRNA-143-A/B/C/D/E/F. All constructs retained functionality as assessed by sensor assays. Shown are statistically significant (adjusted P < 0.05) reductions in normalized relative light units (RLU) in cells transfected with the amplicons (A–F) in comparison to cells transfected with an empty vector (Ø). (c) Sensor assays were performed for an additional nine AmpmiRNA-XX. In each case, sensor activity was decreased in a statistically significant manner (adjusted P < 0.05) compared to experiments involving an empty vector (Ø). (d). The ERK5 protein is a target of miRNA-143. Introduction of AmpmiRNA-143 into HEK 293 T cells was associated with a reduction in ERK5 protein levels as determined by western blot. RNAP II-driven plasmid and an empty vector (Ø) served as positive and negative controls, respectively.
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Figure 4: Functional activity of AmpmiRNA-XX. Luciferase-based miRNA sensor assays were used to compare the functional activity of various miRNA expression units. We created reporter constructs, in which the exact target sequence of a given miRNA was introduced into the 3′UTR of the gene encoding Renilla luciferase and quantified the relative reduction in luciferase levels compared to control experiments involving the same sensor but an empty vector (Ø). An internal firefly luciferase gene served to normalize data. All sensor assays were performed as three independent experiments and data are shown as mean reduction +/– SD compared to control conditions. (a) Functional activity of AmpmiRNA-143, AmpCMV-miRNA-143 and PlaCMV-miRNA-143. Co-transfection of the miRNA-143 sensor and AmpmiRNA-143 led to 57% decrease in luciferase activity, a statistically significant level (P = 0.02) approaching that achieved in transfections using a CMV-driven miRNA-143 expression plasmid (81% reduction) or amplicon (70% reduction) in HEK 293T cells. (b) Functional activity of shorter amplicons AmpmiRNA-143-A/B/C/D/E/F. All constructs retained functionality as assessed by sensor assays. Shown are statistically significant (adjusted P < 0.05) reductions in normalized relative light units (RLU) in cells transfected with the amplicons (A–F) in comparison to cells transfected with an empty vector (Ø). (c) Sensor assays were performed for an additional nine AmpmiRNA-XX. In each case, sensor activity was decreased in a statistically significant manner (adjusted P < 0.05) compared to experiments involving an empty vector (Ø). (d). The ERK5 protein is a target of miRNA-143. Introduction of AmpmiRNA-143 into HEK 293 T cells was associated with a reduction in ERK5 protein levels as determined by western blot. RNAP II-driven plasmid and an empty vector (Ø) served as positive and negative controls, respectively.

Mentions: While quantitative assays revealed the biosynthetic activity of AmpmiRNA-XX, we next sought to assess functional capacity. We created miRNA sensors in which the antisense sequence of a particular mature miRNA was placed in the 3′-UTR of the gene encoding Renilla luciferase. In the presence of mature miRNA, levels of Renilla activity decrease through the classical RNAi pathway. Co-transfection of AmpmiRNA-143 with its sensor led to a statistically significant reduction in Renilla activity, albeit at levels below those attained with CMV-driven miRNA-143 expression vector (Figure 4a). Similar experiments employing the shorter variants AmpmiRNA-143-A/B/C/D/E/F revealed their functional activity as well (Figure 4b). Finally, we used the sensor assay to examine the activity of nine additional AmpmiRNA-XX, all of which were expressed at relatively low levels in HEK 293T cells. In each case, transfection of AmpmiRNA-XX with its sensor led to a statistically significant reduction in Renilla activity (Figure 4c). Next, we asked whether cellular protein levels could be downregulated by exogenously introduced AmpmiRNA-XX. Previous work has identified ERK5 as a target of miRNA-143 (13). Treatment of HEK 293T cells with AmpmiRNA-143 led to the relative reduction of ERK5 protein levels as seen in Figure 4d.Figure 4.


Atypical transcription of microRNA gene fragments.

Song Gao J, Zhang Y, Li M, Tucker LD, Machan JT, Quesenberry P, Rigoutsos I, Ramratnam B - Nucleic Acids Res. (2010)

Functional activity of AmpmiRNA-XX. Luciferase-based miRNA sensor assays were used to compare the functional activity of various miRNA expression units. We created reporter constructs, in which the exact target sequence of a given miRNA was introduced into the 3′UTR of the gene encoding Renilla luciferase and quantified the relative reduction in luciferase levels compared to control experiments involving the same sensor but an empty vector (Ø). An internal firefly luciferase gene served to normalize data. All sensor assays were performed as three independent experiments and data are shown as mean reduction +/– SD compared to control conditions. (a) Functional activity of AmpmiRNA-143, AmpCMV-miRNA-143 and PlaCMV-miRNA-143. Co-transfection of the miRNA-143 sensor and AmpmiRNA-143 led to 57% decrease in luciferase activity, a statistically significant level (P = 0.02) approaching that achieved in transfections using a CMV-driven miRNA-143 expression plasmid (81% reduction) or amplicon (70% reduction) in HEK 293T cells. (b) Functional activity of shorter amplicons AmpmiRNA-143-A/B/C/D/E/F. All constructs retained functionality as assessed by sensor assays. Shown are statistically significant (adjusted P < 0.05) reductions in normalized relative light units (RLU) in cells transfected with the amplicons (A–F) in comparison to cells transfected with an empty vector (Ø). (c) Sensor assays were performed for an additional nine AmpmiRNA-XX. In each case, sensor activity was decreased in a statistically significant manner (adjusted P < 0.05) compared to experiments involving an empty vector (Ø). (d). The ERK5 protein is a target of miRNA-143. Introduction of AmpmiRNA-143 into HEK 293 T cells was associated with a reduction in ERK5 protein levels as determined by western blot. RNAP II-driven plasmid and an empty vector (Ø) served as positive and negative controls, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Functional activity of AmpmiRNA-XX. Luciferase-based miRNA sensor assays were used to compare the functional activity of various miRNA expression units. We created reporter constructs, in which the exact target sequence of a given miRNA was introduced into the 3′UTR of the gene encoding Renilla luciferase and quantified the relative reduction in luciferase levels compared to control experiments involving the same sensor but an empty vector (Ø). An internal firefly luciferase gene served to normalize data. All sensor assays were performed as three independent experiments and data are shown as mean reduction +/– SD compared to control conditions. (a) Functional activity of AmpmiRNA-143, AmpCMV-miRNA-143 and PlaCMV-miRNA-143. Co-transfection of the miRNA-143 sensor and AmpmiRNA-143 led to 57% decrease in luciferase activity, a statistically significant level (P = 0.02) approaching that achieved in transfections using a CMV-driven miRNA-143 expression plasmid (81% reduction) or amplicon (70% reduction) in HEK 293T cells. (b) Functional activity of shorter amplicons AmpmiRNA-143-A/B/C/D/E/F. All constructs retained functionality as assessed by sensor assays. Shown are statistically significant (adjusted P < 0.05) reductions in normalized relative light units (RLU) in cells transfected with the amplicons (A–F) in comparison to cells transfected with an empty vector (Ø). (c) Sensor assays were performed for an additional nine AmpmiRNA-XX. In each case, sensor activity was decreased in a statistically significant manner (adjusted P < 0.05) compared to experiments involving an empty vector (Ø). (d). The ERK5 protein is a target of miRNA-143. Introduction of AmpmiRNA-143 into HEK 293 T cells was associated with a reduction in ERK5 protein levels as determined by western blot. RNAP II-driven plasmid and an empty vector (Ø) served as positive and negative controls, respectively.
Mentions: While quantitative assays revealed the biosynthetic activity of AmpmiRNA-XX, we next sought to assess functional capacity. We created miRNA sensors in which the antisense sequence of a particular mature miRNA was placed in the 3′-UTR of the gene encoding Renilla luciferase. In the presence of mature miRNA, levels of Renilla activity decrease through the classical RNAi pathway. Co-transfection of AmpmiRNA-143 with its sensor led to a statistically significant reduction in Renilla activity, albeit at levels below those attained with CMV-driven miRNA-143 expression vector (Figure 4a). Similar experiments employing the shorter variants AmpmiRNA-143-A/B/C/D/E/F revealed their functional activity as well (Figure 4b). Finally, we used the sensor assay to examine the activity of nine additional AmpmiRNA-XX, all of which were expressed at relatively low levels in HEK 293T cells. In each case, transfection of AmpmiRNA-XX with its sensor led to a statistically significant reduction in Renilla activity (Figure 4c). Next, we asked whether cellular protein levels could be downregulated by exogenously introduced AmpmiRNA-XX. Previous work has identified ERK5 as a target of miRNA-143 (13). Treatment of HEK 293T cells with AmpmiRNA-143 led to the relative reduction of ERK5 protein levels as seen in Figure 4d.Figure 4.

Bottom Line: Here, we report that, in the absence of exogenous promoters, DNA fragments incorporating miRNA precursors can be delivered directly into a variety of human cells and give rise to the corresponding mature miRNA.Notably, the transcription of these miRNA DNA fragments appears resistant to conventional inhibitors of RNAP I/II/III activity.Taken together, our findings suggest the existence of a previously unrecognized atypical transcription program for miRNA precursor sequences.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Rhode Island and Miriam Hospitals, Warren Alpert Medical School, Brown University, Providence, RI 02903, USA.

ABSTRACT
MicroRNAs (miRNAs) are short ( approximately 22 nt) RNAs that impact gene expression by sequence-specific interactions with messenger RNA or promoter sequences of genomic DNA. Ectopic expression of miRNAs can be accomplished by placing fragments of the corresponding miRNA precursor under the control of RNA polymerase II or III (RNAP II/III). Here, we report that, in the absence of exogenous promoters, DNA fragments incorporating miRNA precursors can be delivered directly into a variety of human cells and give rise to the corresponding mature miRNA. Notably, the transcription of these miRNA DNA fragments appears resistant to conventional inhibitors of RNAP I/II/III activity. Taken together, our findings suggest the existence of a previously unrecognized atypical transcription program for miRNA precursor sequences.

Show MeSH
Related in: MedlinePlus