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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 defines transcriptional termination of lnc-pri-miR-122a, b. Mapping nascent transcription across lnc-pri-miR-122 versus GAPDH in Huh7 cells. Gene maps show positions of amplicons used in analysis of chromatin RNA levels (left) and bromo UTP-labeled nuclear run on RNA (right). c. Western blot showing effective depletion of DGCR8 and CPSF-73 by siRNA transfection in Huh7 cells. Error bars represent s.d. of an average (n=3 independent experiments).
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Figure 3: Microprocessor defines transcriptional termination of lnc-pri-miR-122a, b. Mapping nascent transcription across lnc-pri-miR-122 versus GAPDH in Huh7 cells. Gene maps show positions of amplicons used in analysis of chromatin RNA levels (left) and bromo UTP-labeled nuclear run on RNA (right). c. Western blot showing effective depletion of DGCR8 and CPSF-73 by siRNA transfection in Huh7 cells. Error bars represent s.d. of an average (n=3 independent experiments).

Mentions: These results suggested that the lnc-pri-miR-122 3′ end is generated by Drosha cleavage and not by CPA. To characterize the role of the Microprocessor in lnc-pri-miR-122 3′ end formation, we compared the effects of siRNA-mediated knockdown in Huh7 cells of DGCR8 versus the CPA endonuclease CPSF-73. Depletion of both proteins was effective (Fig. 3c). RT-qPCR analysis of RNA isolated from nuclear chromatin, which is enriched in nascent transcripts29, was used to compare profiles across lnc-pri-miRNA-122 and the protein coding gene GAPDH. The level of each RT-qPCR product was normalized to the intron 1 product as a measure of basal nascent transcription, and is shown relative to control siRNA-treated cells. These nascent transcript profiles showed a clear increase in level downstream of the pre-miR-122 sequence in DGCR8 but not CPSF-73 depleted cells (Fig. 3a). We found that Drosha depletion elicited readthrough transcription similar to DGCR8 depletion, while Dicer depletion had no effect (Supplementary Figure 3a). This argues against indirect effects of miRNA depletion on termination and strongly suggests a direct role for the Microprocessor in lnc-pri-miR-122 transcription termination. In contrast, GAPDH nascent transcripts showed no effect of DGCR8 depletion, but as expected showed increased level downstream of the PAS indicating a strong termination defect following CPSF-73 depletion (Fig. 3b). Nuclear run on (NRO) analysis confirmed that DGCR8 knockdown caused a termination defect for lnc-pri-miR-122 but not GAPDH, while CPSF-73 knockdown inhibited termination in GAPDH but not lnc-pri-miR-122 (Fig. 3a,b). Pol II chromatin-immunoprecipitation (ChIP) also confirmed transcriptional readthrough on lnc-pri-miR-122 following DGCR8 depletion (Supplementary Fig. 3b). In sum, we establish that the Microprocessor dictates lnc-pri-miR-122 3′ end formation and transcriptional termination, in contrast to GAPDH which relies on the orthodox CPA complex.


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 defines transcriptional termination of lnc-pri-miR-122a, b. Mapping nascent transcription across lnc-pri-miR-122 versus GAPDH in Huh7 cells. Gene maps show positions of amplicons used in analysis of chromatin RNA levels (left) and bromo UTP-labeled nuclear run on RNA (right). c. Western blot showing effective depletion of DGCR8 and CPSF-73 by siRNA transfection in Huh7 cells. Error bars represent s.d. of an average (n=3 independent experiments).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492989&req=5

Figure 3: Microprocessor defines transcriptional termination of lnc-pri-miR-122a, b. Mapping nascent transcription across lnc-pri-miR-122 versus GAPDH in Huh7 cells. Gene maps show positions of amplicons used in analysis of chromatin RNA levels (left) and bromo UTP-labeled nuclear run on RNA (right). c. Western blot showing effective depletion of DGCR8 and CPSF-73 by siRNA transfection in Huh7 cells. Error bars represent s.d. of an average (n=3 independent experiments).
Mentions: These results suggested that the lnc-pri-miR-122 3′ end is generated by Drosha cleavage and not by CPA. To characterize the role of the Microprocessor in lnc-pri-miR-122 3′ end formation, we compared the effects of siRNA-mediated knockdown in Huh7 cells of DGCR8 versus the CPA endonuclease CPSF-73. Depletion of both proteins was effective (Fig. 3c). RT-qPCR analysis of RNA isolated from nuclear chromatin, which is enriched in nascent transcripts29, was used to compare profiles across lnc-pri-miRNA-122 and the protein coding gene GAPDH. The level of each RT-qPCR product was normalized to the intron 1 product as a measure of basal nascent transcription, and is shown relative to control siRNA-treated cells. These nascent transcript profiles showed a clear increase in level downstream of the pre-miR-122 sequence in DGCR8 but not CPSF-73 depleted cells (Fig. 3a). We found that Drosha depletion elicited readthrough transcription similar to DGCR8 depletion, while Dicer depletion had no effect (Supplementary Figure 3a). This argues against indirect effects of miRNA depletion on termination and strongly suggests a direct role for the Microprocessor in lnc-pri-miR-122 transcription termination. In contrast, GAPDH nascent transcripts showed no effect of DGCR8 depletion, but as expected showed increased level downstream of the PAS indicating a strong termination defect following CPSF-73 depletion (Fig. 3b). Nuclear run on (NRO) analysis confirmed that DGCR8 knockdown caused a termination defect for lnc-pri-miR-122 but not GAPDH, while CPSF-73 knockdown inhibited termination in GAPDH but not lnc-pri-miR-122 (Fig. 3a,b). Pol II chromatin-immunoprecipitation (ChIP) also confirmed transcriptional readthrough on lnc-pri-miR-122 following DGCR8 depletion (Supplementary Fig. 3b). In sum, we establish that the Microprocessor dictates lnc-pri-miR-122 3′ end formation and transcriptional termination, in contrast to GAPDH which relies on the orthodox CPA complex.

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