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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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Ectopically expressed lnc-pri-miR-122 switches to CPA when Microprocessor activity is inhibiteda. Schematic of lnc-pri-miR-122 expression construct driven under HIV-LTR promoter, with locations of northern probe, PCR amplicon and PAS (pA1). WT and Δ plasmids were generated with and without the pre-miR-122 hairpin. b. Northern blot showing that mature miR-122 is expressed from the WT, but not Δ, plasmid following transfection into HeLa cells. U6 snRNA is loading control. c. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the WT plasmid transfected into HeLa cells are the same size as endogenous transcripts in Huh7 cells. d. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the Δ plasmid are larger than the WT transcripts upon transfection into HeLa cells. e. Northern blot showing transcripts generated from the WT plasmid increase in size following Drosha or DGCR8 depletion in HeLa cells. f. RT-qPCR analysis of pA+ or pA− fractionated RNA extracted from siRNA-treated HeLa cells transfected with the WT plasmid. pA+ RNA levels of U6 snRNA, GAPDH mRNA and lnc-pri-miR-122 are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). g. Northern blot showing that mutation of PAS (pA1) does not affect migration of WT lnc-pri-miR-122, but leads to loss of unspliced and spliced transcripts generated from the Δ plasmid upon transfection into HeLa cells.
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Figure 5: Ectopically expressed lnc-pri-miR-122 switches to CPA when Microprocessor activity is inhibiteda. Schematic of lnc-pri-miR-122 expression construct driven under HIV-LTR promoter, with locations of northern probe, PCR amplicon and PAS (pA1). WT and Δ plasmids were generated with and without the pre-miR-122 hairpin. b. Northern blot showing that mature miR-122 is expressed from the WT, but not Δ, plasmid following transfection into HeLa cells. U6 snRNA is loading control. c. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the WT plasmid transfected into HeLa cells are the same size as endogenous transcripts in Huh7 cells. d. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the Δ plasmid are larger than the WT transcripts upon transfection into HeLa cells. e. Northern blot showing transcripts generated from the WT plasmid increase in size following Drosha or DGCR8 depletion in HeLa cells. f. RT-qPCR analysis of pA+ or pA− fractionated RNA extracted from siRNA-treated HeLa cells transfected with the WT plasmid. pA+ RNA levels of U6 snRNA, GAPDH mRNA and lnc-pri-miR-122 are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). g. Northern blot showing that mutation of PAS (pA1) does not affect migration of WT lnc-pri-miR-122, but leads to loss of unspliced and spliced transcripts generated from the Δ plasmid upon transfection into HeLa cells.

Mentions: These results indicated that Pol II transcribing lnc-pri-miR-122 fails to recognize PAS even when Microprocessor cleavage is inhibited. This surprising finding suggested that Pol II might be recruited to the endogenous lnc-pri-miR-122 promoter in a CPA refractory form. To investigate this further, we cloned lnc-pri-miR-122 under the control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter with or without pre-miR-122 hairpin deletion (Fig. 5a). The resulting wild type (WT) and deleted (Δ) plasmids were transfected into HeLa cells, which do not express endogenous lnc-pri-miR-122 (Fig. 1b), together with a plasmid encoding the Tat transcriptional activator. We confirmed that mature miR-122 was expressed from the WT but not Δ plasmid (Fig. 5b). Unspliced and spliced lnc-pri-miR-122 transcripts generated from the WT plasmid were the same size as the endogenous transcripts in Huh7 cells (Fig. 5c), indicating that the site of 3′ end formation is independent of promoter or cell type. Deletion of the pre-miR-122 hairpin or depletion of Drosha or DGCR8 led to production of unspliced and spliced lnc-pri-miR-122 transcripts that migrated at a higher molecular weight than wildtype transcripts (Fig. 5d,e). pA fractionation indicated that lnc-pri-miR-122 RNA generated from the WT plasmid was pA−, similar to U6 snRNA, but became pA+ when DGCR8 was depleted, similar to GAPDH mRNA (Fig. 5f). The 3′ end of Δ RNA was mapped by 3′RACE to a PAS 91nt downstream of the pre-miR-122 hairpin (pA1, Fig 5a). Mutagenesis indicated that this PAS was necessary for 3′ end generation in Δ, but not WT, lnc-pri-miR-122 transcripts (Fig. 5g). This confirmed that in a plasmid context, lnc-pri-miR-122 3′ ends were generated by the Microprocessor, but in the absence of this processing, CPA occurred at a site downstream. Importantly, this was in contrast to the endogenous transcripts, where transcriptional readthrough occurred when Microprocessor cleavage was inhibited, and the PAS was not used (Fig. 3,4).


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

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

Ectopically expressed lnc-pri-miR-122 switches to CPA when Microprocessor activity is inhibiteda. Schematic of lnc-pri-miR-122 expression construct driven under HIV-LTR promoter, with locations of northern probe, PCR amplicon and PAS (pA1). WT and Δ plasmids were generated with and without the pre-miR-122 hairpin. b. Northern blot showing that mature miR-122 is expressed from the WT, but not Δ, plasmid following transfection into HeLa cells. U6 snRNA is loading control. c. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the WT plasmid transfected into HeLa cells are the same size as endogenous transcripts in Huh7 cells. d. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the Δ plasmid are larger than the WT transcripts upon transfection into HeLa cells. e. Northern blot showing transcripts generated from the WT plasmid increase in size following Drosha or DGCR8 depletion in HeLa cells. f. RT-qPCR analysis of pA+ or pA− fractionated RNA extracted from siRNA-treated HeLa cells transfected with the WT plasmid. pA+ RNA levels of U6 snRNA, GAPDH mRNA and lnc-pri-miR-122 are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). g. Northern blot showing that mutation of PAS (pA1) does not affect migration of WT lnc-pri-miR-122, but leads to loss of unspliced and spliced transcripts generated from the Δ plasmid upon transfection into HeLa cells.
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Related In: Results  -  Collection

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Figure 5: Ectopically expressed lnc-pri-miR-122 switches to CPA when Microprocessor activity is inhibiteda. Schematic of lnc-pri-miR-122 expression construct driven under HIV-LTR promoter, with locations of northern probe, PCR amplicon and PAS (pA1). WT and Δ plasmids were generated with and without the pre-miR-122 hairpin. b. Northern blot showing that mature miR-122 is expressed from the WT, but not Δ, plasmid following transfection into HeLa cells. U6 snRNA is loading control. c. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the WT plasmid transfected into HeLa cells are the same size as endogenous transcripts in Huh7 cells. d. Northern blot showing spliced and unspliced lnc-pri-miR-122 transcripts generated from the Δ plasmid are larger than the WT transcripts upon transfection into HeLa cells. e. Northern blot showing transcripts generated from the WT plasmid increase in size following Drosha or DGCR8 depletion in HeLa cells. f. RT-qPCR analysis of pA+ or pA− fractionated RNA extracted from siRNA-treated HeLa cells transfected with the WT plasmid. pA+ RNA levels of U6 snRNA, GAPDH mRNA and lnc-pri-miR-122 are expressed relative to pA− which were set to 1. Error bars represent s.d. of an average (n=3 independent experiments). g. Northern blot showing that mutation of PAS (pA1) does not affect migration of WT lnc-pri-miR-122, but leads to loss of unspliced and spliced transcripts generated from the Δ plasmid upon transfection into HeLa cells.
Mentions: These results indicated that Pol II transcribing lnc-pri-miR-122 fails to recognize PAS even when Microprocessor cleavage is inhibited. This surprising finding suggested that Pol II might be recruited to the endogenous lnc-pri-miR-122 promoter in a CPA refractory form. To investigate this further, we cloned lnc-pri-miR-122 under the control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter with or without pre-miR-122 hairpin deletion (Fig. 5a). The resulting wild type (WT) and deleted (Δ) plasmids were transfected into HeLa cells, which do not express endogenous lnc-pri-miR-122 (Fig. 1b), together with a plasmid encoding the Tat transcriptional activator. We confirmed that mature miR-122 was expressed from the WT but not Δ plasmid (Fig. 5b). Unspliced and spliced lnc-pri-miR-122 transcripts generated from the WT plasmid were the same size as the endogenous transcripts in Huh7 cells (Fig. 5c), indicating that the site of 3′ end formation is independent of promoter or cell type. Deletion of the pre-miR-122 hairpin or depletion of Drosha or DGCR8 led to production of unspliced and spliced lnc-pri-miR-122 transcripts that migrated at a higher molecular weight than wildtype transcripts (Fig. 5d,e). pA fractionation indicated that lnc-pri-miR-122 RNA generated from the WT plasmid was pA−, similar to U6 snRNA, but became pA+ when DGCR8 was depleted, similar to GAPDH mRNA (Fig. 5f). The 3′ end of Δ RNA was mapped by 3′RACE to a PAS 91nt downstream of the pre-miR-122 hairpin (pA1, Fig 5a). Mutagenesis indicated that this PAS was necessary for 3′ end generation in Δ, but not WT, lnc-pri-miR-122 transcripts (Fig. 5g). This confirmed that in a plasmid context, lnc-pri-miR-122 3′ ends were generated by the Microprocessor, but in the absence of this processing, CPA occurred at a site downstream. Importantly, this was in contrast to the endogenous transcripts, where transcriptional readthrough occurred when Microprocessor cleavage was inhibited, and the PAS was not used (Fig. 3,4).

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.

Show MeSH