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Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

Dhir A, Dhir S, Proudfoot NJ, Jopling CL - Nat. Struct. Mol. Biol. (2015)

Bottom Line: We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription.Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes.Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

ABSTRACT
MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

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Microprocessor depletion leads to generation of pA− transcriptional readthrough products on lnc-pri-miR-122a. RT-qPCR analysis of lnc-pri-miR-122 versus U6 snRNA or GAPDH mRNA controls using pA+ or pA− fractionated RNA from siRNA-treated Huh7 cells. pA+ RNA levels are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). b. Chromatin RNA-seq analysis of lnc-pri-miR-122 in control or DGCR8 siRNA-treated Huh7 cells. Red boxed 3′ region is shown below at higher magnification. Position of miRNA (miR-122) is shown by red vertical line.
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Figure 4: Microprocessor depletion leads to generation of pA− transcriptional readthrough products on lnc-pri-miR-122a. RT-qPCR analysis of lnc-pri-miR-122 versus U6 snRNA or GAPDH mRNA controls using pA+ or pA− fractionated RNA from siRNA-treated Huh7 cells. pA+ RNA levels are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). b. Chromatin RNA-seq analysis of lnc-pri-miR-122 in control or DGCR8 siRNA-treated Huh7 cells. Red boxed 3′ region is shown below at higher magnification. Position of miRNA (miR-122) is shown by red vertical line.

Mentions: The transcriptional readthrough we observed following DGCR8 knockdown implied that lnc-pri-miR-122 does not switch to the efficient CPA mechanism of transcriptional termination when the Microprocessor mechanism is inhibited. To address this question directly, we pA-selected RNA from Huh7 cells with or without DGCR8 knockdown. Similarly to U6 snRNA, lnc-pri-miR-122 transcripts remained pA− even when their 3′ end formation was compromised by DGCR8 depletion. In contrast, GAPDH mRNA was strongly pA+ under both conditions (Fig. 4a). By next generation sequencing analysis of chromatin-associated RNA from Huh7 cells (chromatin RNA-seq), we found that DGCR8 depletion leads to extensive transcriptional readthrough for over 5kb downstream of the pre-miR-122 hairpin in lnc-pri-miR-122 (Fig. 4b), despite the presence of several consensus PAS.


Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

Dhir A, Dhir S, Proudfoot NJ, Jopling CL - Nat. Struct. Mol. Biol. (2015)

Microprocessor depletion leads to generation of pA− transcriptional readthrough products on lnc-pri-miR-122a. RT-qPCR analysis of lnc-pri-miR-122 versus U6 snRNA or GAPDH mRNA controls using pA+ or pA− fractionated RNA from siRNA-treated Huh7 cells. pA+ RNA levels are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). b. Chromatin RNA-seq analysis of lnc-pri-miR-122 in control or DGCR8 siRNA-treated Huh7 cells. Red boxed 3′ region is shown below at higher magnification. Position of miRNA (miR-122) is shown by red vertical line.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492989&req=5

Figure 4: Microprocessor depletion leads to generation of pA− transcriptional readthrough products on lnc-pri-miR-122a. RT-qPCR analysis of lnc-pri-miR-122 versus U6 snRNA or GAPDH mRNA controls using pA+ or pA− fractionated RNA from siRNA-treated Huh7 cells. pA+ RNA levels are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). b. Chromatin RNA-seq analysis of lnc-pri-miR-122 in control or DGCR8 siRNA-treated Huh7 cells. Red boxed 3′ region is shown below at higher magnification. Position of miRNA (miR-122) is shown by red vertical line.
Mentions: The transcriptional readthrough we observed following DGCR8 knockdown implied that lnc-pri-miR-122 does not switch to the efficient CPA mechanism of transcriptional termination when the Microprocessor mechanism is inhibited. To address this question directly, we pA-selected RNA from Huh7 cells with or without DGCR8 knockdown. Similarly to U6 snRNA, lnc-pri-miR-122 transcripts remained pA− even when their 3′ end formation was compromised by DGCR8 depletion. In contrast, GAPDH mRNA was strongly pA+ under both conditions (Fig. 4a). By next generation sequencing analysis of chromatin-associated RNA from Huh7 cells (chromatin RNA-seq), we found that DGCR8 depletion leads to extensive transcriptional readthrough for over 5kb downstream of the pre-miR-122 hairpin in lnc-pri-miR-122 (Fig. 4b), despite the presence of several consensus PAS.

Bottom Line: We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription.Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes.Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

ABSTRACT
MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

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