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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-miR-122 transcripts are capped but not polyadenylateda. Gene map showing exon-intron structure and 3′ positioned pre-miR-122 hairpin. Position of primers and Northern probes are indicated. b. Northern blot showing spliced and unspliced lnc-pri-miR-122 from human liver and three cell lines as indicated. Levels of γ-actin mRNA and mature miR-122 were also measured. Note reduced levels of liver RNA were employed. c. Northern blot detecting unspliced and spliced lnc-pri-miR-122 in human liver RNA using intron and exon-specific probes. d. Levels of m7G-cap immunoselected RNA measured by RT-qPCR as indicated. e. Proportion of pA+ RNA relative to pA− for transcripts indicated measured by RT-qPCR, and for lnc-pri-miR-122 and γ-actin measured by northern blotting. Error bars represent s.d. of an average (n=3 independent experiments).
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Figure 1: Lnc-pri-miR-122 transcripts are capped but not polyadenylateda. Gene map showing exon-intron structure and 3′ positioned pre-miR-122 hairpin. Position of primers and Northern probes are indicated. b. Northern blot showing spliced and unspliced lnc-pri-miR-122 from human liver and three cell lines as indicated. Levels of γ-actin mRNA and mature miR-122 were also measured. Note reduced levels of liver RNA were employed. c. Northern blot detecting unspliced and spliced lnc-pri-miR-122 in human liver RNA using intron and exon-specific probes. d. Levels of m7G-cap immunoselected RNA measured by RT-qPCR as indicated. e. Proportion of pA+ RNA relative to pA− for transcripts indicated measured by RT-qPCR, and for lnc-pri-miR-122 and γ-actin measured by northern blotting. Error bars represent s.d. of an average (n=3 independent experiments).

Mentions: First, we characterized the processing of lnc-pri-miR-122 (Fig. 1a). By northern analysis, we identified mature miR-122 and two lnc-pri-miR-122 transcripts of ~4.8 and ~1.9 kilobase (kb) in total RNA from human liver and the human hepatocellular carcinoma cell line Huh7, but not HeLa or HepG2 cells (Fig. 1b). Intron and exon specific probes showed that the larger lnc-pri-miR-122 transcript was unspliced, while the smaller transcript corresponded to inefficiently spliced RNA that lacks an internal 3 kb intron (Fig. 1c). The transcript size indicated that the 3′ end lay close to the pre-miR-122 hairpin, ~2.5kb upstream of a previously identified polyadenylated 3′ end27 (Fig. 1a). We did not detect any longer lnc-pri-miR-122 transcripts. Immunoprecipitation with an antibody directed against the m7G cap demonstrated that lnc-pri-miR-122 was capped, similar to GAPDH mRNA and in agreement with existing CAGE data28 (Fig. 1d). However, quantitative RT-PCR (RT-qPCR) and northern analysis of pA-selected RNA indicated that lnc-pri-miR-122 was non-polyadenylated, in contrast to GAPDH mRNA, but similar to U6 snRNA (Fig. 1e).


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-miR-122 transcripts are capped but not polyadenylateda. Gene map showing exon-intron structure and 3′ positioned pre-miR-122 hairpin. Position of primers and Northern probes are indicated. b. Northern blot showing spliced and unspliced lnc-pri-miR-122 from human liver and three cell lines as indicated. Levels of γ-actin mRNA and mature miR-122 were also measured. Note reduced levels of liver RNA were employed. c. Northern blot detecting unspliced and spliced lnc-pri-miR-122 in human liver RNA using intron and exon-specific probes. d. Levels of m7G-cap immunoselected RNA measured by RT-qPCR as indicated. e. Proportion of pA+ RNA relative to pA− for transcripts indicated measured by RT-qPCR, and for lnc-pri-miR-122 and γ-actin measured by northern blotting. Error bars represent s.d. of an average (n=3 independent experiments).
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Related In: Results  -  Collection

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Figure 1: Lnc-pri-miR-122 transcripts are capped but not polyadenylateda. Gene map showing exon-intron structure and 3′ positioned pre-miR-122 hairpin. Position of primers and Northern probes are indicated. b. Northern blot showing spliced and unspliced lnc-pri-miR-122 from human liver and three cell lines as indicated. Levels of γ-actin mRNA and mature miR-122 were also measured. Note reduced levels of liver RNA were employed. c. Northern blot detecting unspliced and spliced lnc-pri-miR-122 in human liver RNA using intron and exon-specific probes. d. Levels of m7G-cap immunoselected RNA measured by RT-qPCR as indicated. e. Proportion of pA+ RNA relative to pA− for transcripts indicated measured by RT-qPCR, and for lnc-pri-miR-122 and γ-actin measured by northern blotting. Error bars represent s.d. of an average (n=3 independent experiments).
Mentions: First, we characterized the processing of lnc-pri-miR-122 (Fig. 1a). By northern analysis, we identified mature miR-122 and two lnc-pri-miR-122 transcripts of ~4.8 and ~1.9 kilobase (kb) in total RNA from human liver and the human hepatocellular carcinoma cell line Huh7, but not HeLa or HepG2 cells (Fig. 1b). Intron and exon specific probes showed that the larger lnc-pri-miR-122 transcript was unspliced, while the smaller transcript corresponded to inefficiently spliced RNA that lacks an internal 3 kb intron (Fig. 1c). The transcript size indicated that the 3′ end lay close to the pre-miR-122 hairpin, ~2.5kb upstream of a previously identified polyadenylated 3′ end27 (Fig. 1a). We did not detect any longer lnc-pri-miR-122 transcripts. Immunoprecipitation with an antibody directed against the m7G cap demonstrated that lnc-pri-miR-122 was capped, similar to GAPDH mRNA and in agreement with existing CAGE data28 (Fig. 1d). However, quantitative RT-PCR (RT-qPCR) and northern analysis of pA-selected RNA indicated that lnc-pri-miR-122 was non-polyadenylated, in contrast to GAPDH mRNA, but similar to U6 snRNA (Fig. 1e).

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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