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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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Lnc-pri-miRNA may be CPA-incompetent or competent following Microprocessor depletionRNA-seq of pA fractionated nuclear RNA from siRNA-treated HeLa cells. a. Nuclear RNA-seq profile of MIR17HG pA+ and pA− RNA following DGCR8 depletion. b. Nuclear RNA-seq profile of MIRLET7BHG pA+ and pA− RNA following DGCR8 depletion. Position of the PAS (pA) is marked by a dashed vertical line. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and b. c. Model showing CPA in protein coding pri-miRNA allows generation of spliced mRNA and miRNA, while Microprocessor-driven termination in lnc-pri-miRNA generates miRNA and a pA− host transcript that is rapidly degraded. Lnc-pri-miRNA may be CPA-incompetent, leading to transcriptional readthrough and interference when Microprocessor cleavage is inhibited, or CPA-competent allowing effective transcription termination even in the absence of Microprocessor. Red thunderbolt with black fill depicts CPA-mediated cleavage. Blue thunderbolt with red fill depicts Microprocessor-mediated cleavage.
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Figure 8: Lnc-pri-miRNA may be CPA-incompetent or competent following Microprocessor depletionRNA-seq of pA fractionated nuclear RNA from siRNA-treated HeLa cells. a. Nuclear RNA-seq profile of MIR17HG pA+ and pA− RNA following DGCR8 depletion. b. Nuclear RNA-seq profile of MIRLET7BHG pA+ and pA− RNA following DGCR8 depletion. Position of the PAS (pA) is marked by a dashed vertical line. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and b. c. Model showing CPA in protein coding pri-miRNA allows generation of spliced mRNA and miRNA, while Microprocessor-driven termination in lnc-pri-miRNA generates miRNA and a pA− host transcript that is rapidly degraded. Lnc-pri-miRNA may be CPA-incompetent, leading to transcriptional readthrough and interference when Microprocessor cleavage is inhibited, or CPA-competent allowing effective transcription termination even in the absence of Microprocessor. Red thunderbolt with black fill depicts CPA-mediated cleavage. Blue thunderbolt with red fill depicts Microprocessor-mediated cleavage.

Mentions: Similar to endogenous lnc-pri-miR-122, Microprocessor-terminated lnc-pri-miRNA appears to be insensitive to the presence of cryptic PAS, invariably present within their gene and 3′ flanking regions. To further investigate the use of PAS in pri-miRNA, we performed nuclear pA+ and pA− RNA-seq in HeLa cells with or without DGCR8 knockdown. We found that the majority of lnc-pri-miRNA existed as predominantly pA− transcripts, and those that showed extensive readthrough upon loss of Microprocessor remained pA− (Supplementary Table 3). MIR17HG is shown as a specific example (Fig. 8a). It is remarkable that for these Pol II transcripts PAS remain opaque to RNA processing by the CPA complex. However, a few lnc-pri-miRNA utilize PAS to some extent, especially following Microprocessor inactivation. Thus MIRLET7BHG transcripts switched from mainly pA− to pA+ following Microprocessor depletion (Fig. 8b, Supplementary Table 3), and efficient termination occurred at a canonical PAS positioned immediately downstream of pre-miR-let7b. This is similar to the switch to CPA at a downstream PAS that we observed in ectopically expressed lnc-pri-miR-122 (Fig. 5), indicating that this distinction is biologically relevant.


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

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

Lnc-pri-miRNA may be CPA-incompetent or competent following Microprocessor depletionRNA-seq of pA fractionated nuclear RNA from siRNA-treated HeLa cells. a. Nuclear RNA-seq profile of MIR17HG pA+ and pA− RNA following DGCR8 depletion. b. Nuclear RNA-seq profile of MIRLET7BHG pA+ and pA− RNA following DGCR8 depletion. Position of the PAS (pA) is marked by a dashed vertical line. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and b. c. Model showing CPA in protein coding pri-miRNA allows generation of spliced mRNA and miRNA, while Microprocessor-driven termination in lnc-pri-miRNA generates miRNA and a pA− host transcript that is rapidly degraded. Lnc-pri-miRNA may be CPA-incompetent, leading to transcriptional readthrough and interference when Microprocessor cleavage is inhibited, or CPA-competent allowing effective transcription termination even in the absence of Microprocessor. Red thunderbolt with black fill depicts CPA-mediated cleavage. Blue thunderbolt with red fill depicts Microprocessor-mediated cleavage.
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Related In: Results  -  Collection

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Figure 8: Lnc-pri-miRNA may be CPA-incompetent or competent following Microprocessor depletionRNA-seq of pA fractionated nuclear RNA from siRNA-treated HeLa cells. a. Nuclear RNA-seq profile of MIR17HG pA+ and pA− RNA following DGCR8 depletion. b. Nuclear RNA-seq profile of MIRLET7BHG pA+ and pA− RNA following DGCR8 depletion. Position of the PAS (pA) is marked by a dashed vertical line. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and b. c. Model showing CPA in protein coding pri-miRNA allows generation of spliced mRNA and miRNA, while Microprocessor-driven termination in lnc-pri-miRNA generates miRNA and a pA− host transcript that is rapidly degraded. Lnc-pri-miRNA may be CPA-incompetent, leading to transcriptional readthrough and interference when Microprocessor cleavage is inhibited, or CPA-competent allowing effective transcription termination even in the absence of Microprocessor. Red thunderbolt with black fill depicts CPA-mediated cleavage. Blue thunderbolt with red fill depicts Microprocessor-mediated cleavage.
Mentions: Similar to endogenous lnc-pri-miR-122, Microprocessor-terminated lnc-pri-miRNA appears to be insensitive to the presence of cryptic PAS, invariably present within their gene and 3′ flanking regions. To further investigate the use of PAS in pri-miRNA, we performed nuclear pA+ and pA− RNA-seq in HeLa cells with or without DGCR8 knockdown. We found that the majority of lnc-pri-miRNA existed as predominantly pA− transcripts, and those that showed extensive readthrough upon loss of Microprocessor remained pA− (Supplementary Table 3). MIR17HG is shown as a specific example (Fig. 8a). It is remarkable that for these Pol II transcripts PAS remain opaque to RNA processing by the CPA complex. However, a few lnc-pri-miRNA utilize PAS to some extent, especially following Microprocessor inactivation. Thus MIRLET7BHG transcripts switched from mainly pA− to pA+ following Microprocessor depletion (Fig. 8b, Supplementary Table 3), and efficient termination occurred at a canonical PAS positioned immediately downstream of pre-miR-let7b. This is similar to the switch to CPA at a downstream PAS that we observed in ectopically expressed lnc-pri-miR-122 (Fig. 5), indicating that this distinction is biologically relevant.

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
Related in: MedlinePlus