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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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(a) Biosynthetic activity of AmpmiRNA-143 in different human cells. Mature miRNA-143 levels were quantified in amplicon transfected cells and are shown relative to endogenous levels in cells transfected with an empty vector (Ø) with associated 95% CI. All experiments were performed in duplicate, independent experiments. In each cell type, AmpmiRNA-143 increased mature miRNA-143 levels in a statistically significant manner (P < 0.05). (b) Varied cellular activity of amplicons encoding pri-miRNA fragments of miRNA-145, 363 and 517a. All amplicons (252–339 nt) were generated from human genomic DNA and transfected into HEK 293T cells in duplicate, independent experiments. Levels of mature miRNA were quantified by real-time PCR and are expressed relative to background levels in cells transfected with an empty vector (Ø) and 95% CI. Each Amp species led to a statistically significant increase in the levels of the respective miRNA compared to Ø (P < 0.0001). (c) The biosynthetic potential of a given amplicon was quantifiable in cells with relatively low endogenous levels of the miRNA studied. For example, AmpmiRNA-125a significantly (P < 0.0001) increased levels of mature product in Huh-7 cells (miRNA-125alow) but not in HEK 293T cells (miRNA-125ahigh). Conversely, AmpmiRNA-122 produced significantly (P = 0.0008) increased levels of mature product only in HEK 293T cells (miRNA-122low) but not in Huh-7 cells (miRNA-122high). (d) A hybrid amplicon consisting of miRNA-143 and miRNA-125a (AmpmiRNA-125a + 143) was capable of producing both miRNAs at levels comparable to individual expression units after introduction into Huh-7 cells. Relative levels of mature miRNA were quantified by real-time PCR in duplicate experiments and are shown as mean values and 95% CI. All AmpmiRNA-XX constructs including the hybrid molecule were associated with a statistically significant increase (adj P < 0.0001) in the respective mature miRNA levels compared to cells treated with an empty vector (Ø).
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Figure 3: (a) Biosynthetic activity of AmpmiRNA-143 in different human cells. Mature miRNA-143 levels were quantified in amplicon transfected cells and are shown relative to endogenous levels in cells transfected with an empty vector (Ø) with associated 95% CI. All experiments were performed in duplicate, independent experiments. In each cell type, AmpmiRNA-143 increased mature miRNA-143 levels in a statistically significant manner (P < 0.05). (b) Varied cellular activity of amplicons encoding pri-miRNA fragments of miRNA-145, 363 and 517a. All amplicons (252–339 nt) were generated from human genomic DNA and transfected into HEK 293T cells in duplicate, independent experiments. Levels of mature miRNA were quantified by real-time PCR and are expressed relative to background levels in cells transfected with an empty vector (Ø) and 95% CI. Each Amp species led to a statistically significant increase in the levels of the respective miRNA compared to Ø (P < 0.0001). (c) The biosynthetic potential of a given amplicon was quantifiable in cells with relatively low endogenous levels of the miRNA studied. For example, AmpmiRNA-125a significantly (P < 0.0001) increased levels of mature product in Huh-7 cells (miRNA-125alow) but not in HEK 293T cells (miRNA-125ahigh). Conversely, AmpmiRNA-122 produced significantly (P = 0.0008) increased levels of mature product only in HEK 293T cells (miRNA-122low) but not in Huh-7 cells (miRNA-122high). (d) A hybrid amplicon consisting of miRNA-143 and miRNA-125a (AmpmiRNA-125a + 143) was capable of producing both miRNAs at levels comparable to individual expression units after introduction into Huh-7 cells. Relative levels of mature miRNA were quantified by real-time PCR in duplicate experiments and are shown as mean values and 95% CI. All AmpmiRNA-XX constructs including the hybrid molecule were associated with a statistically significant increase (adj P < 0.0001) in the respective mature miRNA levels compared to cells treated with an empty vector (Ø).

Mentions: We next sought to determine whether transcription of AmpmiRNA-143 was restricted to certain cell types only (e.g. HEK 293 T cells). Quantification of mature miRNA-143 in a variety of human cells with relatively low levels of endogenous expression (e.g. PBMC, Huh-7, HeLa) that had been treated with AmpmiRNA-143 readily confirmed its biosynthetic capacity in these cell types as well (Figure 3a). In each case, AmpmiRNA-143 introduction was associated with a statistically significant increase in mature miRNA-143 levels compared to cells that had been transfected with an empty vector.Figure 3.


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)

(a) Biosynthetic activity of AmpmiRNA-143 in different human cells. Mature miRNA-143 levels were quantified in amplicon transfected cells and are shown relative to endogenous levels in cells transfected with an empty vector (Ø) with associated 95% CI. All experiments were performed in duplicate, independent experiments. In each cell type, AmpmiRNA-143 increased mature miRNA-143 levels in a statistically significant manner (P < 0.05). (b) Varied cellular activity of amplicons encoding pri-miRNA fragments of miRNA-145, 363 and 517a. All amplicons (252–339 nt) were generated from human genomic DNA and transfected into HEK 293T cells in duplicate, independent experiments. Levels of mature miRNA were quantified by real-time PCR and are expressed relative to background levels in cells transfected with an empty vector (Ø) and 95% CI. Each Amp species led to a statistically significant increase in the levels of the respective miRNA compared to Ø (P < 0.0001). (c) The biosynthetic potential of a given amplicon was quantifiable in cells with relatively low endogenous levels of the miRNA studied. For example, AmpmiRNA-125a significantly (P < 0.0001) increased levels of mature product in Huh-7 cells (miRNA-125alow) but not in HEK 293T cells (miRNA-125ahigh). Conversely, AmpmiRNA-122 produced significantly (P = 0.0008) increased levels of mature product only in HEK 293T cells (miRNA-122low) but not in Huh-7 cells (miRNA-122high). (d) A hybrid amplicon consisting of miRNA-143 and miRNA-125a (AmpmiRNA-125a + 143) was capable of producing both miRNAs at levels comparable to individual expression units after introduction into Huh-7 cells. Relative levels of mature miRNA were quantified by real-time PCR in duplicate experiments and are shown as mean values and 95% CI. All AmpmiRNA-XX constructs including the hybrid molecule were associated with a statistically significant increase (adj P < 0.0001) in the respective mature miRNA levels compared to cells treated with an empty vector (Ø).
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Figure 3: (a) Biosynthetic activity of AmpmiRNA-143 in different human cells. Mature miRNA-143 levels were quantified in amplicon transfected cells and are shown relative to endogenous levels in cells transfected with an empty vector (Ø) with associated 95% CI. All experiments were performed in duplicate, independent experiments. In each cell type, AmpmiRNA-143 increased mature miRNA-143 levels in a statistically significant manner (P < 0.05). (b) Varied cellular activity of amplicons encoding pri-miRNA fragments of miRNA-145, 363 and 517a. All amplicons (252–339 nt) were generated from human genomic DNA and transfected into HEK 293T cells in duplicate, independent experiments. Levels of mature miRNA were quantified by real-time PCR and are expressed relative to background levels in cells transfected with an empty vector (Ø) and 95% CI. Each Amp species led to a statistically significant increase in the levels of the respective miRNA compared to Ø (P < 0.0001). (c) The biosynthetic potential of a given amplicon was quantifiable in cells with relatively low endogenous levels of the miRNA studied. For example, AmpmiRNA-125a significantly (P < 0.0001) increased levels of mature product in Huh-7 cells (miRNA-125alow) but not in HEK 293T cells (miRNA-125ahigh). Conversely, AmpmiRNA-122 produced significantly (P = 0.0008) increased levels of mature product only in HEK 293T cells (miRNA-122low) but not in Huh-7 cells (miRNA-122high). (d) A hybrid amplicon consisting of miRNA-143 and miRNA-125a (AmpmiRNA-125a + 143) was capable of producing both miRNAs at levels comparable to individual expression units after introduction into Huh-7 cells. Relative levels of mature miRNA were quantified by real-time PCR in duplicate experiments and are shown as mean values and 95% CI. All AmpmiRNA-XX constructs including the hybrid molecule were associated with a statistically significant increase (adj P < 0.0001) in the respective mature miRNA levels compared to cells treated with an empty vector (Ø).
Mentions: We next sought to determine whether transcription of AmpmiRNA-143 was restricted to certain cell types only (e.g. HEK 293 T cells). Quantification of mature miRNA-143 in a variety of human cells with relatively low levels of endogenous expression (e.g. PBMC, Huh-7, HeLa) that had been treated with AmpmiRNA-143 readily confirmed its biosynthetic capacity in these cell types as well (Figure 3a). In each case, AmpmiRNA-143 introduction was associated with a statistically significant increase in mature miRNA-143 levels compared to cells that had been transfected with an empty vector.Figure 3.

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