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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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Deciphering the transcriptional network of AmpmiRNA-143. (a) We assigned RNAP II promoter occupancy to the miRNA-143 gene in HCT116 cells by chromatin immunoprecipitation using antibodies specific to RNAP II. Sheared chromatin from HCT116 cells that had been cross-linked with formaldehyde was immunoprecipitated with anti-RNAP II antibodies. Cross-links were removed and the DNA was purified. The promoter region of miRNA-143 was arbitrarily deconstructed into six segments of ∼100–200 nt each. Specific PCR primers were designed to amplify each component and revealed relative enrichment in sector D, corresponding to nucleotide position –978 with respect to the pre-miRNA start site. All values are relative to nonimmune IgG and normalized to an intergenic control region. Antibodies to GAPDH served as a positive control and revealed ∼30-fold enrichment in the respective promoter region. The corresponding location of AmpmiRNA-143 is shown and lies outside the region of RNAP II enrichment. (b) We used various inhibitors of RNAP to test their effect on the biosynthetic activity of AmpmiRNA-143 as well as RNAP II/III-driven expression constructs. Cells were treated with α-amanitin (50 ug/ml) and actinomycin (2 ug/ml) and transfected with the various constructs prior to harvesting and mature miRNA-143 quantification by real-time PCR. Data are shown as fold reduction in mature miRNA compared to similarly transfected cells in the absence of drug. α-Amanitin significantly reduced the biosynthetic activity of RNAP II (P < 0.001) and III (P = 0.0384)-driven constructs but had no significant effect on the activity of AmpmiRNA-143 (P = 1.0). (c) RNAP II was directly localized to miRNA-143 amplicons harboring the CMV (RNAP II) promoter but not to AmpmiRNA-143 or an amplicon harboring the U6 (RNAP III) promoter. (d) The general transcriptional inhibitor actinomycin reduced the activity of all constructs in a significant manner (P < 0.0001) but its effect on Amp was less pronounced. Data are shown as fold reduction in mature miRNA in drug treated/untreated cells transfected with the indicated constructs. Experiments were performed in duplicate. (e) Effect of RNAP III silencing on AmpmiRNA-143. HEK 293T cells were treated with siRNA against the POLR3A subunit of RNAP III or an irrelevant gene and transfected with either empty vector (Ø), PlaU6-miRNA-143 or AmpmiRNA-143. POLR3A knockdown (∼50–70%) had a significant inhibitory effect (P = 0.003) on the transcription of RNAP III-driven expression vectors but no effect (P = 1.0) on AmpmiRNA-143 as assessed by mature miRNA-143 quantification. Data are shown as fold reduction in mature miRNA compared to cells transfected with an irrelevant siRNA. Experiments were performed in duplicate.
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Figure 5: Deciphering the transcriptional network of AmpmiRNA-143. (a) We assigned RNAP II promoter occupancy to the miRNA-143 gene in HCT116 cells by chromatin immunoprecipitation using antibodies specific to RNAP II. Sheared chromatin from HCT116 cells that had been cross-linked with formaldehyde was immunoprecipitated with anti-RNAP II antibodies. Cross-links were removed and the DNA was purified. The promoter region of miRNA-143 was arbitrarily deconstructed into six segments of ∼100–200 nt each. Specific PCR primers were designed to amplify each component and revealed relative enrichment in sector D, corresponding to nucleotide position –978 with respect to the pre-miRNA start site. All values are relative to nonimmune IgG and normalized to an intergenic control region. Antibodies to GAPDH served as a positive control and revealed ∼30-fold enrichment in the respective promoter region. The corresponding location of AmpmiRNA-143 is shown and lies outside the region of RNAP II enrichment. (b) We used various inhibitors of RNAP to test their effect on the biosynthetic activity of AmpmiRNA-143 as well as RNAP II/III-driven expression constructs. Cells were treated with α-amanitin (50 ug/ml) and actinomycin (2 ug/ml) and transfected with the various constructs prior to harvesting and mature miRNA-143 quantification by real-time PCR. Data are shown as fold reduction in mature miRNA compared to similarly transfected cells in the absence of drug. α-Amanitin significantly reduced the biosynthetic activity of RNAP II (P < 0.001) and III (P = 0.0384)-driven constructs but had no significant effect on the activity of AmpmiRNA-143 (P = 1.0). (c) RNAP II was directly localized to miRNA-143 amplicons harboring the CMV (RNAP II) promoter but not to AmpmiRNA-143 or an amplicon harboring the U6 (RNAP III) promoter. (d) The general transcriptional inhibitor actinomycin reduced the activity of all constructs in a significant manner (P < 0.0001) but its effect on Amp was less pronounced. Data are shown as fold reduction in mature miRNA in drug treated/untreated cells transfected with the indicated constructs. Experiments were performed in duplicate. (e) Effect of RNAP III silencing on AmpmiRNA-143. HEK 293T cells were treated with siRNA against the POLR3A subunit of RNAP III or an irrelevant gene and transfected with either empty vector (Ø), PlaU6-miRNA-143 or AmpmiRNA-143. POLR3A knockdown (∼50–70%) had a significant inhibitory effect (P = 0.003) on the transcription of RNAP III-driven expression vectors but no effect (P = 1.0) on AmpmiRNA-143 as assessed by mature miRNA-143 quantification. Data are shown as fold reduction in mature miRNA compared to cells transfected with an irrelevant siRNA. Experiments were performed in duplicate.

Mentions: We used chromatin precipitation (ChiP) to look for the physical presence of RNAP II on the 2.5-kb genomic region immediately upstream of miRNA-143*. These experiments utilized HCT116 cells that produce high levels of mature miRNA-143. PCR primers amplifying ∼100–200-nt fragments of the putative promoter region were immunoprecipitated with IgG against RNAP II (anti-pol II:8WG16) and revealed RNAP II localization at ∼1 kb upstream relative to the pre-miRNA-143, i.e. in an area well outside of the sequence captured by AmpmiRNA-143 (Figure 5a).Figure 5.


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)

Deciphering the transcriptional network of AmpmiRNA-143. (a) We assigned RNAP II promoter occupancy to the miRNA-143 gene in HCT116 cells by chromatin immunoprecipitation using antibodies specific to RNAP II. Sheared chromatin from HCT116 cells that had been cross-linked with formaldehyde was immunoprecipitated with anti-RNAP II antibodies. Cross-links were removed and the DNA was purified. The promoter region of miRNA-143 was arbitrarily deconstructed into six segments of ∼100–200 nt each. Specific PCR primers were designed to amplify each component and revealed relative enrichment in sector D, corresponding to nucleotide position –978 with respect to the pre-miRNA start site. All values are relative to nonimmune IgG and normalized to an intergenic control region. Antibodies to GAPDH served as a positive control and revealed ∼30-fold enrichment in the respective promoter region. The corresponding location of AmpmiRNA-143 is shown and lies outside the region of RNAP II enrichment. (b) We used various inhibitors of RNAP to test their effect on the biosynthetic activity of AmpmiRNA-143 as well as RNAP II/III-driven expression constructs. Cells were treated with α-amanitin (50 ug/ml) and actinomycin (2 ug/ml) and transfected with the various constructs prior to harvesting and mature miRNA-143 quantification by real-time PCR. Data are shown as fold reduction in mature miRNA compared to similarly transfected cells in the absence of drug. α-Amanitin significantly reduced the biosynthetic activity of RNAP II (P < 0.001) and III (P = 0.0384)-driven constructs but had no significant effect on the activity of AmpmiRNA-143 (P = 1.0). (c) RNAP II was directly localized to miRNA-143 amplicons harboring the CMV (RNAP II) promoter but not to AmpmiRNA-143 or an amplicon harboring the U6 (RNAP III) promoter. (d) The general transcriptional inhibitor actinomycin reduced the activity of all constructs in a significant manner (P < 0.0001) but its effect on Amp was less pronounced. Data are shown as fold reduction in mature miRNA in drug treated/untreated cells transfected with the indicated constructs. Experiments were performed in duplicate. (e) Effect of RNAP III silencing on AmpmiRNA-143. HEK 293T cells were treated with siRNA against the POLR3A subunit of RNAP III or an irrelevant gene and transfected with either empty vector (Ø), PlaU6-miRNA-143 or AmpmiRNA-143. POLR3A knockdown (∼50–70%) had a significant inhibitory effect (P = 0.003) on the transcription of RNAP III-driven expression vectors but no effect (P = 1.0) on AmpmiRNA-143 as assessed by mature miRNA-143 quantification. Data are shown as fold reduction in mature miRNA compared to cells transfected with an irrelevant siRNA. Experiments were performed in duplicate.
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Figure 5: Deciphering the transcriptional network of AmpmiRNA-143. (a) We assigned RNAP II promoter occupancy to the miRNA-143 gene in HCT116 cells by chromatin immunoprecipitation using antibodies specific to RNAP II. Sheared chromatin from HCT116 cells that had been cross-linked with formaldehyde was immunoprecipitated with anti-RNAP II antibodies. Cross-links were removed and the DNA was purified. The promoter region of miRNA-143 was arbitrarily deconstructed into six segments of ∼100–200 nt each. Specific PCR primers were designed to amplify each component and revealed relative enrichment in sector D, corresponding to nucleotide position –978 with respect to the pre-miRNA start site. All values are relative to nonimmune IgG and normalized to an intergenic control region. Antibodies to GAPDH served as a positive control and revealed ∼30-fold enrichment in the respective promoter region. The corresponding location of AmpmiRNA-143 is shown and lies outside the region of RNAP II enrichment. (b) We used various inhibitors of RNAP to test their effect on the biosynthetic activity of AmpmiRNA-143 as well as RNAP II/III-driven expression constructs. Cells were treated with α-amanitin (50 ug/ml) and actinomycin (2 ug/ml) and transfected with the various constructs prior to harvesting and mature miRNA-143 quantification by real-time PCR. Data are shown as fold reduction in mature miRNA compared to similarly transfected cells in the absence of drug. α-Amanitin significantly reduced the biosynthetic activity of RNAP II (P < 0.001) and III (P = 0.0384)-driven constructs but had no significant effect on the activity of AmpmiRNA-143 (P = 1.0). (c) RNAP II was directly localized to miRNA-143 amplicons harboring the CMV (RNAP II) promoter but not to AmpmiRNA-143 or an amplicon harboring the U6 (RNAP III) promoter. (d) The general transcriptional inhibitor actinomycin reduced the activity of all constructs in a significant manner (P < 0.0001) but its effect on Amp was less pronounced. Data are shown as fold reduction in mature miRNA in drug treated/untreated cells transfected with the indicated constructs. Experiments were performed in duplicate. (e) Effect of RNAP III silencing on AmpmiRNA-143. HEK 293T cells were treated with siRNA against the POLR3A subunit of RNAP III or an irrelevant gene and transfected with either empty vector (Ø), PlaU6-miRNA-143 or AmpmiRNA-143. POLR3A knockdown (∼50–70%) had a significant inhibitory effect (P = 0.003) on the transcription of RNAP III-driven expression vectors but no effect (P = 1.0) on AmpmiRNA-143 as assessed by mature miRNA-143 quantification. Data are shown as fold reduction in mature miRNA compared to cells transfected with an irrelevant siRNA. Experiments were performed in duplicate.
Mentions: We used chromatin precipitation (ChiP) to look for the physical presence of RNAP II on the 2.5-kb genomic region immediately upstream of miRNA-143*. These experiments utilized HCT116 cells that produce high levels of mature miRNA-143. PCR primers amplifying ∼100–200-nt fragments of the putative promoter region were immunoprecipitated with IgG against RNAP II (anti-pol II:8WG16) and revealed RNAP II localization at ∼1 kb upstream relative to the pre-miRNA-143, i.e. in an area well outside of the sequence captured by AmpmiRNA-143 (Figure 5a).Figure 5.

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