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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 chromatin RNA-seq profiles across pri-miRNA from HeLa cellsa. Reads (RPKM) across (MIR181A1HG) showing readthrough profiles following Microprocessor depletion. b. Metagene analysis of all expressed lnc-pri-miRNA. c. Reads across a protein coding gene (MCM7) harboring an intronic miRNA cluster showing intron accumulation following Microprocessor depletion d. Metagene analysis of all expressed protein coding genes harboring miRNAs. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and c. Metagene profiles show transcription unit (between transcription start site, TSS and transcription end site, TES) followed by 10 kb of 3′ flanking region in b and d (Mann–Whitney U-test, P < 0.0001, two-tailed, n = 1400, for both cases). e. Western blot showing effective depletion of Drosha and DGCR8 by siRNA transfection in HeLa cells.
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Figure 6: Microprocessor dependent chromatin RNA-seq profiles across pri-miRNA from HeLa cellsa. Reads (RPKM) across (MIR181A1HG) showing readthrough profiles following Microprocessor depletion. b. Metagene analysis of all expressed lnc-pri-miRNA. c. Reads across a protein coding gene (MCM7) harboring an intronic miRNA cluster showing intron accumulation following Microprocessor depletion d. Metagene analysis of all expressed protein coding genes harboring miRNAs. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and c. Metagene profiles show transcription unit (between transcription start site, TSS and transcription end site, TES) followed by 10 kb of 3′ flanking region in b and d (Mann–Whitney U-test, P < 0.0001, two-tailed, n = 1400, for both cases). e. Western blot showing effective depletion of Drosha and DGCR8 by siRNA transfection in HeLa cells.

Mentions: Having demonstrated that chromatin RNA-seq is an effective method of identifying defects in transcriptional termination following Microprocessor depletion (Fig. 4b), we extended this analysis to HeLa cells on a genome-wide scale. HeLa cells were chosen because they express a relatively high number of miRNA. We found that either Drosha or DGCR8 depletion, by siRNA treatment (Fig. 6e), resulted in transcriptional readthrough in most of the expressed lnc-pri-miRNA (Supplementary Table 1). MIR181A1HG and MIR17HG are shown as specific examples (Fig. 6a, Supplementary Fig. 4a), while metagene analysis showed a general termination defect with transcription extending more than 10 kb downstream of lnc-pri-miRNA following Microprocessor depletion (Fig. 6b). A few lnc-pri-miRNA did not shown transcriptional readthrough following Microprocessor depletion (shaded area, Supplementary Table 1); MIRLET7BHG is shown as an example (Supplementary Fig. 4b). Importantly, Dicer knockdown did not affect lnc-pri-miRNA transcriptional termination, confirming that the effects of Microprocessor depletion are direct and not due to loss of mature miRNA (Supplementary Fig. 5).


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 chromatin RNA-seq profiles across pri-miRNA from HeLa cellsa. Reads (RPKM) across (MIR181A1HG) showing readthrough profiles following Microprocessor depletion. b. Metagene analysis of all expressed lnc-pri-miRNA. c. Reads across a protein coding gene (MCM7) harboring an intronic miRNA cluster showing intron accumulation following Microprocessor depletion d. Metagene analysis of all expressed protein coding genes harboring miRNAs. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and c. Metagene profiles show transcription unit (between transcription start site, TSS and transcription end site, TES) followed by 10 kb of 3′ flanking region in b and d (Mann–Whitney U-test, P < 0.0001, two-tailed, n = 1400, for both cases). e. Western blot showing effective depletion of Drosha and DGCR8 by siRNA transfection in HeLa cells.
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Related In: Results  -  Collection

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Figure 6: Microprocessor dependent chromatin RNA-seq profiles across pri-miRNA from HeLa cellsa. Reads (RPKM) across (MIR181A1HG) showing readthrough profiles following Microprocessor depletion. b. Metagene analysis of all expressed lnc-pri-miRNA. c. Reads across a protein coding gene (MCM7) harboring an intronic miRNA cluster showing intron accumulation following Microprocessor depletion d. Metagene analysis of all expressed protein coding genes harboring miRNAs. Direction of transcription indicated by green arrow and positions of miRNA by red vertical lines in a and c. Metagene profiles show transcription unit (between transcription start site, TSS and transcription end site, TES) followed by 10 kb of 3′ flanking region in b and d (Mann–Whitney U-test, P < 0.0001, two-tailed, n = 1400, for both cases). e. Western blot showing effective depletion of Drosha and DGCR8 by siRNA transfection in HeLa cells.
Mentions: Having demonstrated that chromatin RNA-seq is an effective method of identifying defects in transcriptional termination following Microprocessor depletion (Fig. 4b), we extended this analysis to HeLa cells on a genome-wide scale. HeLa cells were chosen because they express a relatively high number of miRNA. We found that either Drosha or DGCR8 depletion, by siRNA treatment (Fig. 6e), resulted in transcriptional readthrough in most of the expressed lnc-pri-miRNA (Supplementary Table 1). MIR181A1HG and MIR17HG are shown as specific examples (Fig. 6a, Supplementary Fig. 4a), while metagene analysis showed a general termination defect with transcription extending more than 10 kb downstream of lnc-pri-miRNA following Microprocessor depletion (Fig. 6b). A few lnc-pri-miRNA did not shown transcriptional readthrough following Microprocessor depletion (shaded area, Supplementary Table 1); MIRLET7BHG is shown as an example (Supplementary Fig. 4b). Importantly, Dicer knockdown did not affect lnc-pri-miRNA transcriptional termination, confirming that the effects of Microprocessor depletion are direct and not due to loss of mature miRNA (Supplementary Fig. 5).

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