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NEAT1 scaffolds RNA-binding proteins and the Microprocessor to globally enhance pri-miRNA processing

Nature Structural & Molecular Biology volume 24, pages 816–824 (2017)Cite this article

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Abstract

MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO–PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha–DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO–PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3′ end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.

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Figure 1: Involvement of paraspeckle-associated proteins and lncRNA in pri-miRNA processing.
Figure 2: Function of NONO–PSF and NEAT1 analyzed with the pri-miRNA-processing reporter.
Figure 3: Genome-wide analysis of NONO–PSF–RNA interactions.
Figure 4: NEAT1 bridges NONO–PSF and the Microprocessor.
Figure 5: NEAT1-mediated interaction networks for enhancing pri-miRNA processing.
Figure 6: Localization of induced pri-miR-1 in paraspeckles in differentiated C2C12 cells and the proposed bird nest model.

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Acknowledgements

This work was supported by grants from the National Key R&D Program of China (2017YFA0504400), the 111 Program of China (B06018), NIH (HG004659, GM049369, GM052872), the National Natural Science Foundation of China (31000573), and the Postdoctoral Science Foundation of China (20090451074).

Author information

Author notes

  1. Xiang-Dong Fu

    Present address: Institute of Genomic Medicine, University of California, San Diego, La Jolla, California, USA

  2. Qi-Jia Wu

    Present address: Seqhealth Technology Co., Ltd, Wuhan, China

  3. Yi Zhang

    Present address: ABLife Inc., Wuhan, China

  4. Li Jiang, Changwei Shao and Qi-Jia Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Virology and Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China

    Li Jiang, Changwei Shao, Qi-Jia Wu, Geng Chen, Jie Zhou, Bo Yang, Yi Zhang, Yu Zhou & Xiang-Dong Fu

  2. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA

    Changwei Shao, Hairi Li, Lan-Tao Gou, Gene W Yeo & Xiang-Dong Fu

  3. Institute of Molecular Medicine, Peking University, Beijing, China

    Yangming Wang

  4. Institute for Advanced Studies, Wuhan University, Wuhan, China

    Yu Zhou

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Contributions

Q.-J.W., L.J., C.S., and X.-D.F. designed the experiments. L.J., Q.-J.W., and C.S. performed most experiments. J.Z., B.Y., and Y. Zhou analyzed the data; G.C., H.L., L.-T.G., Y. Zhang, Y.W. and G.W.Y. contributed to sequencing, cell lines, and data interpretation. L.J., C.S., Y. Zhou and X.-D.F. wrote the paper.

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Correspondence to Yu Zhou or Xiang-Dong Fu.

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Integrated supplementary information

Supplementary Figure 1 Differential expression of miRNAs from the pri-miR-17-92a locus.

(a) The miR-17-92a expression unit in the third intron of the host miR-17HG transcript. (b,c) Impact of knockdown of Ago1-4 on miRNA expression from the miR-17-92a locus, quantified by RT-qPCR (b) and the Ago knockdown efficiency was verified by Western blotting (c). (d,e) Impact of knockdown of Dicer on miRNA expression from the miR-17-92a locus, quantified by RT-qPCR (d) and the Dicer knockdown efficiency verified by Western blotting (e). Uncropped images of Western blots in c and e are shown in Supplementary Data Set 1. Data in b,d are presented as mean ± SEM (n=3, technical replicates). *P < 0.05; **P < 0.01; ***P < 0.001, determined by two-tailed Student’s t test. Source data are reported in Source Data for Supplementary Figure 1.

Source data

Supplementary Figure 2 Evidence for the involvement of paraspeckle-associated proteins and NEAT1 in miRNA biogenesis.

(a) The knockdown efficiencies of PSF (left) and NONO (right) were verified by Western blotting. (b,c) Ablation of paraspeckle key components PSF (b) or NONO (c) down regulated representative miRNAs as indicated. (d) Illustration of miRNA sensor reporters: Individual antisense miRNAs were cloned into the 3’UTR of the Renilla luciferase in psiCHECK2. Reduced miRNA expression would stabilize the Renilla mRNA, thus increasing the relative luciferase activity. (e) Levels of miRNA sensor reporters in response to knockdown of individual paraspeckle-associated proteins or NEAT1. GFP and the RNA binding protein PTB1 served as controls. (f) Knockout of PSPC1 by CRISPR up-regulates representative miRNAs as indicated. (g) The absence of PSPC1 was verified by Western blotting in two independent cell lines. Uncropped images of Western blots in a,g are shown in Supplementary Data Set 1. Data in b,c,e,f are presented as mean ± SEM (n=3, technical replicates for b,c,f; n=4, cell culture for e). *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, determined by two-tailed Student’s t test. Source data are reported in Source Data for Supplementary Figure 2.

Source data

Supplementary Figure 3 Interrelationship of paraspeckle associated proteins and RNA.

(a) HeLa cells were immunostained with anti-NONO (green) and anti-PSPC1 (red) antibodies. DAPI was used to indicate nucleus. In wild-type cells (WT), NONO and PSPC1 co-localized in paraspeckles. In response to knockdown (KD) of PSF (second raw) or NONO (third raw), the paraspeckle marker PSCP1 no longer showed foci. In contrast, in PSPC1 knockout (KO) cells, NONO continued to show foci. Scale bars, 10 μm. (b) RT-qPCR was performed to confirm NEAT1 knockdown with a stealth siRNA and the impact on miRNA expression. (c-f) Western blot analysis of the Microprocessor Drosha/DGCR8 and a panel of other paraspeckle-associated RBPs upon knockdown of paraspeckle-associated proteins or NEAT1. Data in b are presented as mean ± SEM (n=3, technical replicates). *P < 0.05; **P < 0.01; ***P < 0.001, determined by two-tailed Student’s t test. Source data are reported in Source Data for Supplementary Figure 3. Uncropped images of Western blots are shown in Supplementary Data Set 1.

Source data

Supplementary Figure 4 miRNA profiling by small RNA-seq and validation by RT-qPCR.

(a,b) Counts of reference sequences (tRNAs, snoRNAs, spike-in RNA, rRNAs) between duplicated small RNA-seq libraries from siGFP-treated cells (a) or between siPSF and siGFP-treated cells (b). (c,d) Correlation of miRNA counts between duplicated libraries from siGFP-treated (c) or siPSF-treated HeLa cells (d). (e) A panel of miRNAs validated by RT-qPCR. (f) Heatmap representation of validated miRNA expression from the miR-17-92a locus (left) or other pri-miRNAs (right) in response to knockdown of various paraspeckle-associated proteins or NEAT1 as indicated. Data in e are presented as mean ± SEM (n=3, technical replicates). **P < 0.01; ***P < 0.001; NS, not significant, determined by two-tailed Student’s t test. Data source data are reported in Source Data for Supplementary Figure 4.

Source data

Supplementary Figure 5 CLIP-seq analysis of PSF/NONO interactions with RNA.

(a) The anti-PSF immunoprecipitated complex trimmed with two different concentrations of MNase (1: 1,000 or 1: 50,000 dilution) and analyzed by autoradiography (bottom panel) and Western blotting (upper panel). (b) Reproducibility between independently constructed CLIP-seq libraries for NONO and PSF. Global comparison was performed with 1 Kb-binned genome. (c) The genomic distribution of NONO and PSF CLIP-seq peaks. (d) Additional examples of NONO and PSF binding on a set of pri-miRNAs in HeLa cell. Uncropped images of Western blots and autoradiography in a are shown in Supplementary Data Set 1.

Supplementary Figure 6 Exogenous DGCR8 interacts with endogenous NONO.

(a) The pri-miRNA-based reporter assay for pri-miR-612 processing relative to pri-miR-17-92a processing in response to knockdown of the Microprocessor. (b) Detection of FLAG-tagged DGCR8 in anti-NONO immunoprecipitant. (c) Detection of NONO in FLAG-tagged DGCR8 immunoprecipitant. * indicates IgG heavy chain. Data are shown in a as mean ± SEM (n=3, cell culture). *P < 0.05; ***P < 0.001; NS, not significant, determined by two-tailed Student’s t test. Data source are reported in Source Data for Supplementary Figure 6. Uncropped images of Western blots in b and c are shown in Supplementary Data Set 1.

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6. (PDF 1283 kb)

Life Sciences Reporting Summary (PDF 129 kb)

Supplementary Table 1

DNA and RNA oligos used in this study. (XLSX 16 kb)

Supplementary Table 2

All the antibodies used in this study. (XLSX 10 kb)

Supplementary Table 3

Peptides identified by mass spectrometry. (XLSX 22 kb)

Supplementary Table 4

Deep sequencing statistics. (XLSX 9 kb)

Supplementary Data Set 1

PDF files for all western blotting original gel images. (PDF 8296 kb)

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Jiang, L., Shao, C., Wu, QJ. et al. NEAT1 scaffolds RNA-binding proteins and the Microprocessor to globally enhance pri-miRNA processing. Nat Struct Mol Biol 24, 816–824 (2017). https://doi.org/10.1038/nsmb.3455

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