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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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Activity of miRNA-like amplicons. (a) We created two AmpmiRNA-XX variants in which the mature and (*) sequences of miRNA 143 and 30a were replaced by those encoding the guide and passenger strand of siRNA targeting the HIV-1 transactivator (tat) protein. (b) Northern blot was used to validate the activity of AmpmiRNA-143/tat. When miRNA-143/tat was placed under the direction of RNAP III (U6), we could detect tat siRNA. However, when the construct was introduced as a promoter-less AmpmiRNA-143/tat, tat siRNA could not be detected. Similar results were obtained for Amp miRNA-30a/tat.(c, d) The failure of Amp miRNA-xx/tat prompted us to manipulate the backbone of miRNA-143 by replacing its mature and (*) sequences with those encoding miRNA-145. Real-time PCR was used to quantify mature miRNA-145 levels in cells that had been transfected with either empty vector (Ø) or AmpmiRNA-143/145. Amp species were associated with a statistically significant 1000-fold increase (P < 0.001) in mature miRNA-145 levels compared to cells that had been treated with Ø. (d). miRNA-145 sensor assays were used to quantify the activity of AmpmiRNA-143/145. Compared to a U6-driven construct, Amp miRNA-143/145 reduced sensor activity in a statistically significant manner (P < 0.05).
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Figure 8: Activity of miRNA-like amplicons. (a) We created two AmpmiRNA-XX variants in which the mature and (*) sequences of miRNA 143 and 30a were replaced by those encoding the guide and passenger strand of siRNA targeting the HIV-1 transactivator (tat) protein. (b) Northern blot was used to validate the activity of AmpmiRNA-143/tat. When miRNA-143/tat was placed under the direction of RNAP III (U6), we could detect tat siRNA. However, when the construct was introduced as a promoter-less AmpmiRNA-143/tat, tat siRNA could not be detected. Similar results were obtained for Amp miRNA-30a/tat.(c, d) The failure of Amp miRNA-xx/tat prompted us to manipulate the backbone of miRNA-143 by replacing its mature and (*) sequences with those encoding miRNA-145. Real-time PCR was used to quantify mature miRNA-145 levels in cells that had been transfected with either empty vector (Ø) or AmpmiRNA-143/145. Amp species were associated with a statistically significant 1000-fold increase (P < 0.001) in mature miRNA-145 levels compared to cells that had been treated with Ø. (d). miRNA-145 sensor assays were used to quantify the activity of AmpmiRNA-143/145. Compared to a U6-driven construct, Amp miRNA-143/145 reduced sensor activity in a statistically significant manner (P < 0.05).

Mentions: Our experiments suggested that the miRNA species examined above possessed either sequence and/or structural determinants that enabled the recruitment of an unconventional transcriptional machinery. To gain mechanistic insight into the perplexing nature of atypical transcription, we first compared the nucleotide sequence of the various miRNA associated with functional amplicons and could not identify a conserved sequence motif. All amplicons, however, shared two major attributes upon transcription: their hairpin-like RNA structure and subsequent stereotypic processing by RNAase III enzymes into shorter fragments of mature and (*) miRNA and possibly other 21–22-nt fragments, such as the recently characterized miRNA-offset RNA (23,24). We reasoned that as long as the hairpin structure and Dicer/Drosha processing sites were maintained, the primary sequence of any given AmpmiRNA-XX species could be altered yet remain functional. Previously, we and others have demonstrated that the miRNA backbone can be modified to include sequences of a given siRNA and thereby serve as efficient vectors of delivery upon incorporation into RNAP III-driven expression cassettes (25). Thus, we introduced sequences encoding a previously validated siRNA targeting HIV-1 tat into the backbone of miRNA-143 (Figure 8a). Unexpectedly, when miRNA-143/tat was PCR-amplified and introduced as a promoter-less Amp, we could not detect tat siRNA expression. However, introduction of an RNAP III-driven expression cassette harboring miRNA-143/tat into HEK 293T cells led to tat shRNA detection, as assessed by northern blot (Figure 8b). Thus, the sequence replacement did not prevent the molecule’s entry into and processing by the cellular RNAi machinery upon placement of the construct under the direction of a strong promoter. Similar negative results were encountered when tat siRNA sequences were embedded in miRNA-30a (data not shown). The only seeming distinction between functional AmpmiRNA-XX versus non-functional AmpmiRNA-/tat was the replacement of mature and (*) miRNA sequences with those encoding the tat siRNA guide and passenger strand, respectively. It was entirely unclear to us why engineered miRNA/tat molecules failed to undergo atypical transcription when delivered as Amp. We next modified the miRNA-143 backbone such that mature and (*) sequences were replaced with those encoding miRNA-145. Introduction of this hybrid molecule led to the production of mature, functional miRNA-145 in a statistically significant manner (Figure 8c and d). Thus, alteration of the miRNA backbone in and of itself does not appear to impact the biosynthetic potential of Amp as much as the choice of cargo.Figure 8.


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)

Activity of miRNA-like amplicons. (a) We created two AmpmiRNA-XX variants in which the mature and (*) sequences of miRNA 143 and 30a were replaced by those encoding the guide and passenger strand of siRNA targeting the HIV-1 transactivator (tat) protein. (b) Northern blot was used to validate the activity of AmpmiRNA-143/tat. When miRNA-143/tat was placed under the direction of RNAP III (U6), we could detect tat siRNA. However, when the construct was introduced as a promoter-less AmpmiRNA-143/tat, tat siRNA could not be detected. Similar results were obtained for Amp miRNA-30a/tat.(c, d) The failure of Amp miRNA-xx/tat prompted us to manipulate the backbone of miRNA-143 by replacing its mature and (*) sequences with those encoding miRNA-145. Real-time PCR was used to quantify mature miRNA-145 levels in cells that had been transfected with either empty vector (Ø) or AmpmiRNA-143/145. Amp species were associated with a statistically significant 1000-fold increase (P < 0.001) in mature miRNA-145 levels compared to cells that had been treated with Ø. (d). miRNA-145 sensor assays were used to quantify the activity of AmpmiRNA-143/145. Compared to a U6-driven construct, Amp miRNA-143/145 reduced sensor activity in a statistically significant manner (P < 0.05).
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Figure 8: Activity of miRNA-like amplicons. (a) We created two AmpmiRNA-XX variants in which the mature and (*) sequences of miRNA 143 and 30a were replaced by those encoding the guide and passenger strand of siRNA targeting the HIV-1 transactivator (tat) protein. (b) Northern blot was used to validate the activity of AmpmiRNA-143/tat. When miRNA-143/tat was placed under the direction of RNAP III (U6), we could detect tat siRNA. However, when the construct was introduced as a promoter-less AmpmiRNA-143/tat, tat siRNA could not be detected. Similar results were obtained for Amp miRNA-30a/tat.(c, d) The failure of Amp miRNA-xx/tat prompted us to manipulate the backbone of miRNA-143 by replacing its mature and (*) sequences with those encoding miRNA-145. Real-time PCR was used to quantify mature miRNA-145 levels in cells that had been transfected with either empty vector (Ø) or AmpmiRNA-143/145. Amp species were associated with a statistically significant 1000-fold increase (P < 0.001) in mature miRNA-145 levels compared to cells that had been treated with Ø. (d). miRNA-145 sensor assays were used to quantify the activity of AmpmiRNA-143/145. Compared to a U6-driven construct, Amp miRNA-143/145 reduced sensor activity in a statistically significant manner (P < 0.05).
Mentions: Our experiments suggested that the miRNA species examined above possessed either sequence and/or structural determinants that enabled the recruitment of an unconventional transcriptional machinery. To gain mechanistic insight into the perplexing nature of atypical transcription, we first compared the nucleotide sequence of the various miRNA associated with functional amplicons and could not identify a conserved sequence motif. All amplicons, however, shared two major attributes upon transcription: their hairpin-like RNA structure and subsequent stereotypic processing by RNAase III enzymes into shorter fragments of mature and (*) miRNA and possibly other 21–22-nt fragments, such as the recently characterized miRNA-offset RNA (23,24). We reasoned that as long as the hairpin structure and Dicer/Drosha processing sites were maintained, the primary sequence of any given AmpmiRNA-XX species could be altered yet remain functional. Previously, we and others have demonstrated that the miRNA backbone can be modified to include sequences of a given siRNA and thereby serve as efficient vectors of delivery upon incorporation into RNAP III-driven expression cassettes (25). Thus, we introduced sequences encoding a previously validated siRNA targeting HIV-1 tat into the backbone of miRNA-143 (Figure 8a). Unexpectedly, when miRNA-143/tat was PCR-amplified and introduced as a promoter-less Amp, we could not detect tat siRNA expression. However, introduction of an RNAP III-driven expression cassette harboring miRNA-143/tat into HEK 293T cells led to tat shRNA detection, as assessed by northern blot (Figure 8b). Thus, the sequence replacement did not prevent the molecule’s entry into and processing by the cellular RNAi machinery upon placement of the construct under the direction of a strong promoter. Similar negative results were encountered when tat siRNA sequences were embedded in miRNA-30a (data not shown). The only seeming distinction between functional AmpmiRNA-XX versus non-functional AmpmiRNA-/tat was the replacement of mature and (*) miRNA sequences with those encoding the tat siRNA guide and passenger strand, respectively. It was entirely unclear to us why engineered miRNA/tat molecules failed to undergo atypical transcription when delivered as Amp. We next modified the miRNA-143 backbone such that mature and (*) sequences were replaced with those encoding miRNA-145. Introduction of this hybrid molecule led to the production of mature, functional miRNA-145 in a statistically significant manner (Figure 8c and d). Thus, alteration of the miRNA backbone in and of itself does not appear to impact the biosynthetic potential of Amp as much as the choice of cargo.Figure 8.

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