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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 dependent termination prevents transcriptional interferencea. Chromatin RNA-seq profiles across MIR17HG-GPC5 tandem gene locus showing readthrough transcription following Microprocessor depletion. Black arrow and red box highlights reduction in GPC5 exon 1 peak following Microprocessor depletion. b. RT-qPCR analysis of chimeric transcripts versus GPC5 exon 1. Gene specific RT primer (arrowhead) in exon 1 and PCR amplicons are indicated by black bars. c. Chromatin RNA-seq profiles across convergent OGFRL1-LINC00472 gene locus. d. Read quantification for protein coding genes subject to transcriptional interference following Microprocessor depletion. e. mRNA levels of GPC5 and OGFRL1 were determined by RT-qPCR using exon specific primers. RNA levels are expressed relative to control siRNA which were set to 1. All values are normalized to GAPDH mRNA. f. Protein levels of GPC5 and OGFRL1 are reduced by transcriptional interference. Direction of transcription is indicated by green arrow and miRNA by red vertical lines. All experiments used HeLa cells. Error bars represent s.d. of an average (n=3 independent experiments).
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Figure 7: Microprocessor dependent termination prevents transcriptional interferencea. Chromatin RNA-seq profiles across MIR17HG-GPC5 tandem gene locus showing readthrough transcription following Microprocessor depletion. Black arrow and red box highlights reduction in GPC5 exon 1 peak following Microprocessor depletion. b. RT-qPCR analysis of chimeric transcripts versus GPC5 exon 1. Gene specific RT primer (arrowhead) in exon 1 and PCR amplicons are indicated by black bars. c. Chromatin RNA-seq profiles across convergent OGFRL1-LINC00472 gene locus. d. Read quantification for protein coding genes subject to transcriptional interference following Microprocessor depletion. e. mRNA levels of GPC5 and OGFRL1 were determined by RT-qPCR using exon specific primers. RNA levels are expressed relative to control siRNA which were set to 1. All values are normalized to GAPDH mRNA. f. Protein levels of GPC5 and OGFRL1 are reduced by transcriptional interference. Direction of transcription is indicated by green arrow and miRNA by red vertical lines. All experiments used HeLa cells. Error bars represent s.d. of an average (n=3 independent experiments).

Mentions: We identified specific examples of lnc-pri-miRNA genes in which the transcriptional readthrough induced by Microprocessor depletion extended into a downstream protein coding gene, either in convergent or tandem orientation. We reasoned that such readthrough transcription might downregulate the invaded gene by a transcriptional interference mechanism31. For the tandem MIR17HG-GPC5 locus, Microprocessor depletion caused the MIR17HG transcript to extend over 20 kb, reading into GPC5 (Fig. 7a, Supplementary Fig. 8). Chimeric transcripts were readily detected, as was a substantial reduction in GPC5 exon 1 RNA levels (Fig. 7b, d, Supplementary Fig. 8). Both GPC5 mRNA and protein levels were more than 70% reduced (Fig. 7e,f), indicating a clear transcriptional interference effect caused by loss of Microprocessor-mediated termination. For the convergent OGFRL1-LINC00472 locus, loss of Microprocessor caused LINC00472 transcripts to read through into OGFRL1, again causing transcriptional down-regulation (Fig. 7c,d). OGFRL1 mRNA levels dropped 80% while protein levels were 50% lower (Fig. 7e,f). Possibly this protein has higher stability than GPC5. These data imply that convergent transcription can also induce gene inactivation, possibly by Pol II collision effects32. Notably Dicer depletion had no effect on GPC5 or OGFRL1 mRNA level (Fig. 7e), indicating that the effects of DGCR8 knockdown are due to transcriptional interference and not miRNA-mediated mRNA destabilization.


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 dependent termination prevents transcriptional interferencea. Chromatin RNA-seq profiles across MIR17HG-GPC5 tandem gene locus showing readthrough transcription following Microprocessor depletion. Black arrow and red box highlights reduction in GPC5 exon 1 peak following Microprocessor depletion. b. RT-qPCR analysis of chimeric transcripts versus GPC5 exon 1. Gene specific RT primer (arrowhead) in exon 1 and PCR amplicons are indicated by black bars. c. Chromatin RNA-seq profiles across convergent OGFRL1-LINC00472 gene locus. d. Read quantification for protein coding genes subject to transcriptional interference following Microprocessor depletion. e. mRNA levels of GPC5 and OGFRL1 were determined by RT-qPCR using exon specific primers. RNA levels are expressed relative to control siRNA which were set to 1. All values are normalized to GAPDH mRNA. f. Protein levels of GPC5 and OGFRL1 are reduced by transcriptional interference. Direction of transcription is indicated by green arrow and miRNA by red vertical lines. All experiments used HeLa cells. Error bars represent s.d. of an average (n=3 independent experiments).
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Figure 7: Microprocessor dependent termination prevents transcriptional interferencea. Chromatin RNA-seq profiles across MIR17HG-GPC5 tandem gene locus showing readthrough transcription following Microprocessor depletion. Black arrow and red box highlights reduction in GPC5 exon 1 peak following Microprocessor depletion. b. RT-qPCR analysis of chimeric transcripts versus GPC5 exon 1. Gene specific RT primer (arrowhead) in exon 1 and PCR amplicons are indicated by black bars. c. Chromatin RNA-seq profiles across convergent OGFRL1-LINC00472 gene locus. d. Read quantification for protein coding genes subject to transcriptional interference following Microprocessor depletion. e. mRNA levels of GPC5 and OGFRL1 were determined by RT-qPCR using exon specific primers. RNA levels are expressed relative to control siRNA which were set to 1. All values are normalized to GAPDH mRNA. f. Protein levels of GPC5 and OGFRL1 are reduced by transcriptional interference. Direction of transcription is indicated by green arrow and miRNA by red vertical lines. All experiments used HeLa cells. Error bars represent s.d. of an average (n=3 independent experiments).
Mentions: We identified specific examples of lnc-pri-miRNA genes in which the transcriptional readthrough induced by Microprocessor depletion extended into a downstream protein coding gene, either in convergent or tandem orientation. We reasoned that such readthrough transcription might downregulate the invaded gene by a transcriptional interference mechanism31. For the tandem MIR17HG-GPC5 locus, Microprocessor depletion caused the MIR17HG transcript to extend over 20 kb, reading into GPC5 (Fig. 7a, Supplementary Fig. 8). Chimeric transcripts were readily detected, as was a substantial reduction in GPC5 exon 1 RNA levels (Fig. 7b, d, Supplementary Fig. 8). Both GPC5 mRNA and protein levels were more than 70% reduced (Fig. 7e,f), indicating a clear transcriptional interference effect caused by loss of Microprocessor-mediated termination. For the convergent OGFRL1-LINC00472 locus, loss of Microprocessor caused LINC00472 transcripts to read through into OGFRL1, again causing transcriptional down-regulation (Fig. 7c,d). OGFRL1 mRNA levels dropped 80% while protein levels were 50% lower (Fig. 7e,f). Possibly this protein has higher stability than GPC5. These data imply that convergent transcription can also induce gene inactivation, possibly by Pol II collision effects32. Notably Dicer depletion had no effect on GPC5 or OGFRL1 mRNA level (Fig. 7e), indicating that the effects of DGCR8 knockdown are due to transcriptional interference and not miRNA-mediated mRNA destabilization.

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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