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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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3′ end mapping, subcellular distribution and rapid turnover of lnc-pri-miR-122a. Gene map of lnc-pri-miR-122 showing position of primers for RT-qPCR and 3′ end mapping. b. PolyA polymerase (PAP)-dependent 3′ end mapping using 3′ RACE. Position of 3′ RACE PCR product shown by gel fractionation and location of mapped 3′ end cleavage products are shown by red arrow heads on the pre-miR-122 hairpin structure (see Supplementary Fig. 2). c. Pri-miR-122 and GAPDH mRNA distribution were determined between cell fractions (WC denotes whole cell, N nuclear and C cytoplasmic) by RT-PCR using indicated primers. Western blot shows purity of nuclear and cytoplasmic fractions by use of cytoplasmic and nuclear specific protein antibodies. d. RNA stability following actinomycin D inhibition of transcription at various time points measured by RT-qPCR of lnc-pri-miR-122 transcripts versus GAPDH mRNA. RNA levels are expressed relative to the levels at time=0, which were set to 1. Huh7 cells were used in all experiments. Error bars represent s.d. of an average (n=3 independent experiments).
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Figure 2: 3′ end mapping, subcellular distribution and rapid turnover of lnc-pri-miR-122a. Gene map of lnc-pri-miR-122 showing position of primers for RT-qPCR and 3′ end mapping. b. PolyA polymerase (PAP)-dependent 3′ end mapping using 3′ RACE. Position of 3′ RACE PCR product shown by gel fractionation and location of mapped 3′ end cleavage products are shown by red arrow heads on the pre-miR-122 hairpin structure (see Supplementary Fig. 2). c. Pri-miR-122 and GAPDH mRNA distribution were determined between cell fractions (WC denotes whole cell, N nuclear and C cytoplasmic) by RT-PCR using indicated primers. Western blot shows purity of nuclear and cytoplasmic fractions by use of cytoplasmic and nuclear specific protein antibodies. d. RNA stability following actinomycin D inhibition of transcription at various time points measured by RT-qPCR of lnc-pri-miR-122 transcripts versus GAPDH mRNA. RNA levels are expressed relative to the levels at time=0, which were set to 1. Huh7 cells were used in all experiments. Error bars represent s.d. of an average (n=3 independent experiments).

Mentions: To map lnc-pri-miR-122 3′ ends at nucleotide resolution, we developed a pA tail-independent 3′RACE technique. We mapped lnc-pri-miR-122 3′ ends to just upstream of the site of Drosha cleavage on the 5′ arm of the pre-miR-122 hairpin. We observed some heterogeneity of 3′ end location, presumably due to exonucleolytic trimming following Drosha cleavage (Fig. 2b, Supplementary Fig. 2). Both unspliced and spliced lnc-pri-miR-122 were retained in the nucleus and rapidly degraded (Fig. 2c,d), as expected for non-polyadenylated transcripts.


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

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

3′ end mapping, subcellular distribution and rapid turnover of lnc-pri-miR-122a. Gene map of lnc-pri-miR-122 showing position of primers for RT-qPCR and 3′ end mapping. b. PolyA polymerase (PAP)-dependent 3′ end mapping using 3′ RACE. Position of 3′ RACE PCR product shown by gel fractionation and location of mapped 3′ end cleavage products are shown by red arrow heads on the pre-miR-122 hairpin structure (see Supplementary Fig. 2). c. Pri-miR-122 and GAPDH mRNA distribution were determined between cell fractions (WC denotes whole cell, N nuclear and C cytoplasmic) by RT-PCR using indicated primers. Western blot shows purity of nuclear and cytoplasmic fractions by use of cytoplasmic and nuclear specific protein antibodies. d. RNA stability following actinomycin D inhibition of transcription at various time points measured by RT-qPCR of lnc-pri-miR-122 transcripts versus GAPDH mRNA. RNA levels are expressed relative to the levels at time=0, which were set to 1. Huh7 cells were used in all experiments. Error bars represent s.d. of an average (n=3 independent experiments).
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Figure 2: 3′ end mapping, subcellular distribution and rapid turnover of lnc-pri-miR-122a. Gene map of lnc-pri-miR-122 showing position of primers for RT-qPCR and 3′ end mapping. b. PolyA polymerase (PAP)-dependent 3′ end mapping using 3′ RACE. Position of 3′ RACE PCR product shown by gel fractionation and location of mapped 3′ end cleavage products are shown by red arrow heads on the pre-miR-122 hairpin structure (see Supplementary Fig. 2). c. Pri-miR-122 and GAPDH mRNA distribution were determined between cell fractions (WC denotes whole cell, N nuclear and C cytoplasmic) by RT-PCR using indicated primers. Western blot shows purity of nuclear and cytoplasmic fractions by use of cytoplasmic and nuclear specific protein antibodies. d. RNA stability following actinomycin D inhibition of transcription at various time points measured by RT-qPCR of lnc-pri-miR-122 transcripts versus GAPDH mRNA. RNA levels are expressed relative to the levels at time=0, which were set to 1. Huh7 cells were used in all experiments. Error bars represent s.d. of an average (n=3 independent experiments).
Mentions: To map lnc-pri-miR-122 3′ ends at nucleotide resolution, we developed a pA tail-independent 3′RACE technique. We mapped lnc-pri-miR-122 3′ ends to just upstream of the site of Drosha cleavage on the 5′ arm of the pre-miR-122 hairpin. We observed some heterogeneity of 3′ end location, presumably due to exonucleolytic trimming following Drosha cleavage (Fig. 2b, Supplementary Fig. 2). Both unspliced and spliced lnc-pri-miR-122 were retained in the nucleus and rapidly degraded (Fig. 2c,d), as expected for non-polyadenylated transcripts.

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