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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

(a) Schema showing various sized amplicons (#1–#4) of AmpmiRNA-517a with and without the RNAP III specific A and B boxes. (b) Relative amount of mature miRNA-517a produced upon cellular introduction of the various constructs was quantified by real-time PCR as compared to cells transfected with an empty vector (Ø). While A and B box inclusive amplicons produced the highest levels of mature miRNA, the 206-bp amplicon without A/B box retained the ability to produce mature miRNA. Experiments were performed in duplicate and constructs #1–#4 all produced significantly higher levels of mature product than Ø (P < 0.05). (c) The functionality of AmpmiRNA-517a was assessed by sensor assays. While the RNAP III (U6)-driven expression vector retained the most potency (∼80%), shorter amplicons with and without A/B boxes also reduced Renilla relative light units by ∼30–40%. All constructs significantly inhibited sensor activity (P < 0.005). (d) Effect of silencing RNAP III on AmpmiRNA-517a amplicons. Cells were treated with anti-POLR3A or an irrelevant siRNA and transfected with amplicons prior to harvesting and quantifying mature miRNA-517a levels. RNAP III knockdown had no effect on AmpmiRNA-517a biosynthetic activity (P = 0.17) yet significantly decreased the synthetic capacity of both RNAP III-driven expression vector (P = 0.0006) and amplicons harboring A/B boxes (P = 0.0001).
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Figure 6: (a) Schema showing various sized amplicons (#1–#4) of AmpmiRNA-517a with and without the RNAP III specific A and B boxes. (b) Relative amount of mature miRNA-517a produced upon cellular introduction of the various constructs was quantified by real-time PCR as compared to cells transfected with an empty vector (Ø). While A and B box inclusive amplicons produced the highest levels of mature miRNA, the 206-bp amplicon without A/B box retained the ability to produce mature miRNA. Experiments were performed in duplicate and constructs #1–#4 all produced significantly higher levels of mature product than Ø (P < 0.05). (c) The functionality of AmpmiRNA-517a was assessed by sensor assays. While the RNAP III (U6)-driven expression vector retained the most potency (∼80%), shorter amplicons with and without A/B boxes also reduced Renilla relative light units by ∼30–40%. All constructs significantly inhibited sensor activity (P < 0.005). (d) Effect of silencing RNAP III on AmpmiRNA-517a amplicons. Cells were treated with anti-POLR3A or an irrelevant siRNA and transfected with amplicons prior to harvesting and quantifying mature miRNA-517a levels. RNAP III knockdown had no effect on AmpmiRNA-517a biosynthetic activity (P = 0.17) yet significantly decreased the synthetic capacity of both RNAP III-driven expression vector (P = 0.0006) and amplicons harboring A/B boxes (P = 0.0001).

Mentions: Further support for the lack of involvement of RNAP III in AmpmiRNA-XX transcription derived from more detailed experiments involving miRNA-517a. As mentioned, AmpmiRNA-517a was associated with mature miRNA production after introduction into HEK 293T cells. The genetic organization of miRNA-517a is somewhat unique in that it is a substrate for RNAP III with a concise organization including the traditional A and B boxes required for RNAP III docking (Figure 6a) (4). Importantly, our initial AmpmiRNA-517a was 262 nt in length and did not contain either of the known A or B boxes located upstream of its pre-miRNA sequence. We created a series of progressively longer variants of the original AmpmiRNA-517a, the longest of which contained both the A and B boxes. Interestingly, segmental truncations of AmpmiR-517a, with partial or complete removal of the A and B box motifs, also led to detectable levels of functional mature miRNA-517a, albeit at levels far lower than the two amplicon variants (#3 and #4) that included both boxes (Figure 6b and c). Not surprisingly, cells treated with a siRNA targeting POLR3A exhibited a severely compromised ability to transcribe the miRNA-517a variants that contained either or both of the A and B boxes. However, in the presence of the siRNA targeting POLR3A, the same cells retained fully their ability to transcribe the shorter AmpmiRNA-517a that lacked the A and B boxes (Figure 6d). Thus, although wild-type miRNA-517a is under the control of RNAP III through the A and B boxes, an amplicon that does not include A and B boxes appears to operate upon transfection in a manner similar to the other amplicons we described earlier.Figure 6.


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) Schema showing various sized amplicons (#1–#4) of AmpmiRNA-517a with and without the RNAP III specific A and B boxes. (b) Relative amount of mature miRNA-517a produced upon cellular introduction of the various constructs was quantified by real-time PCR as compared to cells transfected with an empty vector (Ø). While A and B box inclusive amplicons produced the highest levels of mature miRNA, the 206-bp amplicon without A/B box retained the ability to produce mature miRNA. Experiments were performed in duplicate and constructs #1–#4 all produced significantly higher levels of mature product than Ø (P < 0.05). (c) The functionality of AmpmiRNA-517a was assessed by sensor assays. While the RNAP III (U6)-driven expression vector retained the most potency (∼80%), shorter amplicons with and without A/B boxes also reduced Renilla relative light units by ∼30–40%. All constructs significantly inhibited sensor activity (P < 0.005). (d) Effect of silencing RNAP III on AmpmiRNA-517a amplicons. Cells were treated with anti-POLR3A or an irrelevant siRNA and transfected with amplicons prior to harvesting and quantifying mature miRNA-517a levels. RNAP III knockdown had no effect on AmpmiRNA-517a biosynthetic activity (P = 0.17) yet significantly decreased the synthetic capacity of both RNAP III-driven expression vector (P = 0.0006) and amplicons harboring A/B boxes (P = 0.0001).
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Figure 6: (a) Schema showing various sized amplicons (#1–#4) of AmpmiRNA-517a with and without the RNAP III specific A and B boxes. (b) Relative amount of mature miRNA-517a produced upon cellular introduction of the various constructs was quantified by real-time PCR as compared to cells transfected with an empty vector (Ø). While A and B box inclusive amplicons produced the highest levels of mature miRNA, the 206-bp amplicon without A/B box retained the ability to produce mature miRNA. Experiments were performed in duplicate and constructs #1–#4 all produced significantly higher levels of mature product than Ø (P < 0.05). (c) The functionality of AmpmiRNA-517a was assessed by sensor assays. While the RNAP III (U6)-driven expression vector retained the most potency (∼80%), shorter amplicons with and without A/B boxes also reduced Renilla relative light units by ∼30–40%. All constructs significantly inhibited sensor activity (P < 0.005). (d) Effect of silencing RNAP III on AmpmiRNA-517a amplicons. Cells were treated with anti-POLR3A or an irrelevant siRNA and transfected with amplicons prior to harvesting and quantifying mature miRNA-517a levels. RNAP III knockdown had no effect on AmpmiRNA-517a biosynthetic activity (P = 0.17) yet significantly decreased the synthetic capacity of both RNAP III-driven expression vector (P = 0.0006) and amplicons harboring A/B boxes (P = 0.0001).
Mentions: Further support for the lack of involvement of RNAP III in AmpmiRNA-XX transcription derived from more detailed experiments involving miRNA-517a. As mentioned, AmpmiRNA-517a was associated with mature miRNA production after introduction into HEK 293T cells. The genetic organization of miRNA-517a is somewhat unique in that it is a substrate for RNAP III with a concise organization including the traditional A and B boxes required for RNAP III docking (Figure 6a) (4). Importantly, our initial AmpmiRNA-517a was 262 nt in length and did not contain either of the known A or B boxes located upstream of its pre-miRNA sequence. We created a series of progressively longer variants of the original AmpmiRNA-517a, the longest of which contained both the A and B boxes. Interestingly, segmental truncations of AmpmiR-517a, with partial or complete removal of the A and B box motifs, also led to detectable levels of functional mature miRNA-517a, albeit at levels far lower than the two amplicon variants (#3 and #4) that included both boxes (Figure 6b and c). Not surprisingly, cells treated with a siRNA targeting POLR3A exhibited a severely compromised ability to transcribe the miRNA-517a variants that contained either or both of the A and B boxes. However, in the presence of the siRNA targeting POLR3A, the same cells retained fully their ability to transcribe the shorter AmpmiRNA-517a that lacked the A and B boxes (Figure 6d). Thus, although wild-type miRNA-517a is under the control of RNAP III through the A and B boxes, an amplicon that does not include A and B boxes appears to operate upon transfection in a manner similar to the other amplicons we described earlier.Figure 6.

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