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Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs

Nature Structural & Molecular Biology volume 22, pages 319–327 (2015)Cite this article

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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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Figure 1: lnc-pri-miR-122 transcripts are capped but not polyadenylated.
Figure 2: 3′-end mapping, subcellular distribution and rapid turnover of lnc-pri-miR-122.
Figure 3: Microprocessor defines transcriptional termination of lnc-pri-miR-122.
Figure 4: Microprocessor depletion leads to generation of pA− transcriptional readthrough products on lnc-pri-miR-122.
Figure 5: Ectopically expressed lnc-pri-miR-122 switches to CPA when Microprocessor activity is inhibited.
Figure 6: Microprocessor-dependent chromatin RNA–seq profiles across pri-miRNAs from HeLa cells.
Figure 7: Microprocessor-dependent termination prevents transcriptional interference.
Figure 8: lnc-pri-miRNA may be CPA incompetent or competent after Microprocessor depletion.

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  • 12 March 2015

    In the version of this article initially published online, the title 'Microprocessor mediates transcriptional termination in genes encoding long noncoding microRNAs' should have read 'Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs'. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Proudfoot laboratory for advice and encouragement. This work was supported by a Programme grant (091805/Z/10/Z) from the Wellcome Trust, a European Research Council Advanced Award (339270-polyloop) to N.J.P. and a Biotechnology and Biological Sciences Research Council David Phillips Fellowship (BB/F02360X/1) to C.L.J. High-throughput sequencing was performed by the Genomics group at The Oxford Wellcome Centre for Human Genetics.

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Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Ashish Dhir, Somdutta Dhir & Nick J Proudfoot

  2. School of Pharmacy, University of Nottingham, Nottingham, UK

    Catherine L Jopling

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  1. Ashish Dhir
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Contributions

A.D. and C.L.J. performed molecular biology experiments; S.D. performed bioinformatics analysis; A.D., N.J.P. and C.L.J. designed the experiments and wrote the manuscript.

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Correspondence to Nick J Proudfoot or Catherine L Jopling.

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Integrated supplementary information

Supplementary Figure 1 Distribution of miRNAs between lncRNA and protein-coding genes.

Pie chart showing distribution of human miRNA between protein coding and lncRNA genes. To the right, lncRNA miRNA genes are further subdivided into intergenic (lincRNA) or other less well-characterized subdivisions such as pseudogene or antisense.

Supplementary Figure 2 Mapped 3′ ends of lnc-pri-miR-122.

Sequencing results of lnc-pri-miR-122 3’RACE products amplified by poly(A) polymerase dependent method. Red arrow head marks the 3’ end cleavage sites based on sequence analysis.

Supplementary Figure 3 Microprocessor but not Dicer is required for lnc-pri-miRNA-122 transcription termination.

a. Chromatin-associated Huh7 RNA was analyzed by RT-qPCR as in Fig. 3a. b. Pol II ChIP and qPCR analysis of lnc-pri-miR-122 following DGCR8 depletion in Huh7 cells. Positions of the primers are indicated on the gene map above. Error bars represent s.d. of an average (n=3 independent experiments).

Supplementary Figure 4 Microprocessor depletion leads to transcriptional readthrough on MIR17HG but not MIRLET7BHG.

a. Chromatin RNA-seq profile for the MIR17HG locus following Drosha or DGCR8 depletion in HeLa cells. b. Chromatin RNA-seq profile for the MIRLET7BHG locus following Drosha or DGCR8 depletion.

Supplementary Figure 5 Dicer depletion does not lead to transcriptional readthrough on lnc-pri-miRNA.

a. Western blot showing effective Dicer depletion by siRNA transfection in HeLa cells. b. Chromatin RNA-seq profiles for MIR181A1HG and LINC00472 following DGCR8 or Dicer depletion in HeLa cells.

Supplementary Figure 6 Effect of Microprocessor knockdown on levels of TSS transcripts in genes containing miRNAs in HeLa cells.

a. and b. TSS metagene plot of chromatin RNA-seq of lnc-pri-miRNA versus protein coding genes harboring miRNA showing region

1 kb before and after TSS. TSS denotes transcription start site.

Supplementary Figure 7 Scatter plots showing reproducibility of replicate chromatin RNA–seq in HeLa cells.

a. Replicate of control siRNA treated samples. b. Replicate of DGCR8 siRNA treated samples.

Supplementary Figure 8 Additional views of GPC5 chromatin RNA–seq profiles.

a. Compressed view showing full extent of MIR17HG-GPC5 transcription unit. b. Magnified view of GPC5 exon 1 following Microprocessor knockdown in HeLa cells. Coding sequence (CDS) that translates first 55 amino acids of the GPC5 protein is located in exon 1 and is denoted by bracket. HeLa cell RNA employed. Direction of transcription indicated by green arrows.

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Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–6 (PDF 1572 kb)

Supplementary Data Set 1

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Dhir, A., Dhir, S., Proudfoot, N. et al. Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs. Nat Struct Mol Biol 22, 319–327 (2015). https://doi.org/10.1038/nsmb.2982

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