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Exon-intron circular RNAs regulate transcription in the nucleus

Nature Structural & Molecular Biology volume 22, pages 256–264 (2015)Cite this article

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A Corrigendum to this article was published on 06 February 2017

This article has been updated

Abstract

Noncoding RNAs (ncRNAs) have numerous roles in development and disease, and one of the prominent roles is to regulate gene expression. A vast number of circular RNAs (circRNAs) have been identified, and some have been shown to function as microRNA sponges in animal cells. Here, we report a class of circRNAs associated with RNA polymerase II in human cells. In these circRNAs, exons are circularized with introns 'retained' between exons; we term them exon-intron circRNAs or EIciRNAs. EIciRNAs predominantly localize in the nucleus, interact with U1 snRNP and promote transcription of their parental genes. Our findings reveal a new role for circRNAs in regulating gene expression in the nucleus, in which EIciRNAs enhance the expression of their parental genes in cis, and highlight a regulatory strategy for transcriptional control via specific RNA-RNA interaction between U1 snRNA and EIciRNAs.

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Figure 1: Identification and characterization of EIciRNA.
Figure 2: Sequences related to circularization.
Figure 3: EIciRNAs have cis regulatory effects.
Figure 4: Double FISH for circRNA and its parental or neighboring gene loci.
Figure 5: EIciRNAs interact with Pol II, U1 snRNP and parental gene promoters.
Figure 6: U1 snRNP is indispensable for the cis effects of EIciRNAs.
Figure 7: RNA-RNA interaction between U1 snRNA and EIciRNAs is essential for the effect of EIciRNAs.
Figure 8: A working model for the cis effects of EIciRNAs on the expression of parental genes.

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Change history

  • 19 August 2016

    In the version of this article initially published, the convergent primers depicted in the schematic in Figure 1b were incorrectly placed. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors thank S. Altman, Y. Shi, R. Chen, E. Wang, W.W. Walthall, P. Jin, S. Guang, X. Song, Q. Liu, Y. Mei and M. Wu for discussions and members of the Shan laboratory for discussions and technical support. This work was supported by grants to G.S. (the National Basic Research Program of China, 2011CBA01103 and 2015CB943000; the National Natural Science Foundation of China, 81171074, 91232702 and 31471225; the Chinese Academy of Sciences, 1731112304041 and KJZD-EW-L01-2; and the Fundamental Research Funds for the Central Universities of China, WK2070000034), Z.L. (the National Natural Science Foundation of China, 81372215 and 31301069) and Y.J. (the National Natural Science Foundation of China, 11175068).

Author information

Author notes

  1. Zhaoyong Li, Chuan Huang and Chun Bao: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, University of Science and Technology of China, Hefei, China

    Zhaoyong Li, Chuan Huang, Chun Bao, Liang Chen, Mei Lin, Xiaolin Wang, Guolin Zhong, Bin Yu, Wanchen Hu, Limin Dai, Pengfei Zhu, Zhaoxia Chang, Qingfa Wu, Huijie Liu & Ge Shan

  2. Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, University of Science and Technology of China, Hefei, China

    Zhaoyong Li & Ge Shan

  3. Department of Physics, Central China Normal University, Wuhan, China

    Chun Bao & Ya Jia

  4. Institute of Biophysics, Central China Normal University, Wuhan, China

    Chun Bao & Ya Jia

  5. Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China

    Yi Zhao

  6. National Center for Protein Sciences, Beijing, China

    Ping Xu

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Contributions

G.S. conceived this project, designed experiments and supervised their execution. G.S. wrote the manuscript with the assistance of Z.L. and C.H. Z.L., C.H. and C.B. performed most of the experiments and analyzed most of the data. L.C., M.L., X.W., G.Z., B.Y., W.H., L.D., Y.J., P.X. and H.L. performed some of the experiments and (or) data analysis. X.W., P.Z., Z.C., Q.W. and Y.Z. performed bioinformatic analysis. All authors discussed the results and made comments on the manuscript.

Corresponding author

Correspondence to Ge Shan.

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Integrated supplementary information

Supplementary Figure 1 EIciRNAs were pulled down with an antibody to Pol II.

(a) Western blot showing that pol II was efficiently pulled down with a pol II antibody in HeLa cell lysates. (b) The enrichment of 15 circRNAs in pol II CLIP was verified by qRT-PCR together with known pol II-associated U1 snRNA in HeLa cells. U7 snRNA was used as a negative control. CDR1as is a circular RNA and known microRNA sponge, which showed enrichment in Ago2 CLIP but not in pol II CLIP. (c)The same experiment as in b was performed with HEK293 cells, and the data also showed enrichment for circRNAs in pol II CLIP. (d) Pol II RNA IP followed with DNase digestion treatment demonstrated that the association of circRNAs with pol II was abolished, indicating that pull down of EIciRNAs by pol II is mediated by genomic DNA i.e., both pol II and EIciRNAs might bind to chromatin for interaction. (e) RT-PCR gels show that CDR1as is enriched but circEIF3J and circPAIP2 is not in Ago2 CLIP. (f) RT-PCR gels show that circCLTC is expressed in HeLa cells but not in HEK293 cells although CLTC mRNA was expressed in both HeLa and HEK293 cells. RT-PCR primers (black arrows) for CLTC mRNA and circCLTC are indicated beneath the gel. *, P value < 0.05, **, P value < 0.01. P values were determined with the two-tailed Student’s t-test. All data were from triplicate experiments. Error bars represent the s.e.m.

Supplementary Figure 2 Verification of circRNAs with RT-PCR and FISH.

(a) RT-PCR was performed with divergent primers corresponding to intron (primer set 1) or exon (primer set 2) sequences in circEIF3J and circPAIP2. PCR products show that introns were retained in circEIF3J and circPAIP2. (b) RT-PCR was performed with divergent primers corresponding to the 5’ exon and upstream intron closest to the 3’ exon. PCR products show that at least the upstream intron closest to the 3’ exon was retained in all of the other 13 circRNAs. (c) Separate channels are shown for FISH images for circEIF3J and circPAIP2 in Fig. 1f. From these and subsequent FISH images, it is clear that circEIF3J and circPAIP2 are localized in the nucleus, but both also have some cellular heterogeneity in the nuclear distribution and perhaps copy number. (d) Separate channels are shown for the FISH images of EIF3J and PAIP2 mRNA in Fig. 1f. (e) Double FISH of the parental gene loci and a downstream intron of the indicated circRNA in the parental gene pre-mRNA. Essentially no FISH signal of the downstream intron was observed for the downstream intron. For EIF3J, only one (indicated with a white arrowhead) out of 38 cells imaged showed a positive FISH signal for the downstream intron (n=38 cells for EIF3J, and 30 cells for PAIP2). (f) FISH Images of circRNAs with or without siRNA knockdown of the indicated circRNA. (g) Dual FISH of circRNAs and mRNA of the parental genes after treatment with RNase R. No FISH signal was detected for the mRNA. For all FISH images for circRNAs, junction probes were used unless specified. All FISH images were with HEK293 cells.

Supplementary Figure 3 Overexpression of circRNA and effects of EIciRNA or mRNA knockdown.

(a) Gel images show the size of the RT-PCR products with the divergent primers. Except for the vector control and the circ-Exon constructs, the overexpressed circRNAs were also intron retaining examined with the “primer set 1”. The RT-PCR bands show the size of the PCR product, and the band intensity does not represent relative expression levels. The junction in the circRNA produced by the circEIF3J_1 kb plasmid was different from that of circEIF3J, and this difference is shown in the gel image and sequence diagram below. (b) Knockdown of circEIF3J or circPAIP2 with the siRNA resulted in a decrease in the parental gene mRNA level in HEK293 cells. (c) ASO sequences for the knockdown of circEIF3J and circPAIP2. circEIF3J ASO was used to target the intron contained in circEIF3J, and a control ASO targeted intron 4 in the EIF3J gene; circPAIP2 ASO targeted the junction of circPAIP2, and a scrambled ASO was used as a control. Sequences in black indicated DNA, and sequences in green indicate RNA. (d) Knockdown of circEIF3J had no effect on the mRNA level of PAIP2 and CTDSPL2 (the 5’ neighbouring gene of EIF3J); knockdown of circPAIP2 had no effect on the mRNA level of EIF3J or MATR3 (the 5’ neighbouring gene of PAIP2). (e) siRNA knockdown efficiency of circEIF3J and circPAIP2 for the experiments shown in Fig 3c. (f) Nuclear run-on experiments showed that mRNA knockdown had no effect on the transcription of the indicated genes. siRNA knockdown efficiency of EIF3J or PAIP2 mRNA is shown below the nuclear run-on data. All data were from triplicate experiments. Error bars represent the s.e.m. **, P value < 0.01. P values were determined with two-tailed Student’s t-test.

Supplementary Figure 4 Effect of parental gene knockdown and circRNA overexpression.

(a-c) Knockdown of EIF3J or PAIP2 mRNA with shRNA plasmids or siRNA had no effect on the level of circRNA. The diagrams below the bar figures show the positions of the shRNA- or siRNA-targeted sites in EIF3J or PAIP2. (d and e) Overexpression of circEIF3J or circPAIP2 with plasmids had no effect on the level of EIF3J or PAIP2 mRNA in HeLa (d) and HEK293 cells (e). All data were from triplicate experiments. Error bars represent the s.e.m. *, P value < 0.05, **, P value < 0.01. P values were determined with the two-tailed Student’s t-test.

Supplementary Figure 5 EIciRNA pulldown.

(a) Biotin-labeled oligos (circEIF3J oligo and circPAIP2 oligo) complementary to the junction and intron sequences of the circRNAs were used for pulldown experiments. Oligos complementary to a downstream exon were used as control (ciE-con and ciP-con). circEIF3J and circPAIP2 could be pulled down by circRNA oligos as demonstrated by comparison with control oligos. The pulldown efficiency of the circRNAs and U7 snRNA (using biotin-labeled oligos complementary to U7) as percentage of input is shown in the right panel. Pre-mRNA of EIF3J and PAIP2 was not detected (N. D.) in the pulldown materials as examined with primer pairs corresponding to the last intron and exon of the gene. Semi-quantitative RT-PCR gels for circRNA (115 bp amplicon for circEIF3J, and 166 bp circPAIP2) and parental gene mRNA (97 bp amplicon for EIF3J mRNA, and 147 bp PAIP2) from the pulldown experiments are shown below. (b) Enrichment of U1, U2, U4, U5, and U6 snRNA in pol II CLIP was verified by qRT-PCR using HeLa cells. U7 snRNA and 5s rRNA were used as negative controls. (c) RT-PCR gel of U2 snRNA co-precipitated with circRNA. (d) Interactions between molecules were crosslinked, pol II was pre-cleared by immunoprecipitation (7.7% pol II remained), and pulldown of the circRNAs still yielded a significant amount of U1 snRNA compared with a pulldown without pol II pre-clearing. This result indicates that the interaction between U1 snRNA and circEIF3J or circPAIP2 does not necessarily require the presence of pol II. In contrast, it appears that most, if not all, U2 snRNA may interact with circRNA indirectly via association with pol II. (e and f) The pulldown experiments were carried out with Biotin-labeled oligos against the exonic sequences in the EIciRNA (ciEIF3J-E oligo and ciPAIP2-E oligo); Oligos complementary to downstream exons in the mRNA were used as control (ciE-con-E and ciP-con-E). Similar results were obtained as compared to data with exon-intron oligos, although the pulldown efficiency of ciEIF3J-E oligo and ciPAIP2-E oligo was lower. N.D. (not detected, CT>35). All data were from triplicate experiments. Error bars represent the s.e.m. **, P value < 0.01. P values were determined with a two-tailed Student’s t-test.

Supplementary Figure 6 ChIRP of circRNA.

(a) PCR gels show that CHIRP of the indicated circRNAs could pull down the promoters of corresponding parent genes (up panel) but not the promoters of the corresponding neighbouring genes (lower panel). pEIF3J (B), the B site shown in Fig. 3d was examined; pPAIP2 (D), the D site shown in Fig. 3d was examined. Promoter sites for CTDSPL2 and MATR3 are shown in the diagram below the gel images. (b) Generally, gene bodies corresponding to parent genes could not be pulled down with CHIRP. One site for each gene body (D’ site for EIF3J; G’ site for PAIP2) showed some enrichment although to lesser degree compared with the enrichment found at the promoter (Fig. 5d). D’ for EIF3J and G’ for PAIP2 are sites in the large introns just upstream of the circRNA sequences. Sites examined in the gene body are shown below the bar figures. Error bars represent the s.e.m. * P value < 0.05, P values were determined with a two-tailed Student’s t-test. (c) ChIP results using the U1A, U1C, U2AF35, or U2AF65 antibodies for the promoter of the neighbouring genes (CTDSPL2 for EIF3J and MATR3 for PAIP2) are shown as a comparison for Fig. 3f. These results indicate that U1 snRNP (U1A & U1C) binds to promoters of genes such as EIF3J and PAIP2 but not to the promoters of CTDSPL2 and MATR3. All data were from triplicate experiments.

Supplementary Figure 7 FISH images of circRNA, U1 snRNA and parental gene loci.

(a) Additional representative Dual FISH images of circRNA and U1 snRNA. The Y/G ratio (Y refers to the yellow area, which is the colocalisation signal of circRNA and U1 snRNA; G refers to the green area and U1 snRNA signal) and the Y/R ratio (R refers to the red area and the circRNA signal) were calculated for each cell, and the data shown in Fig. 3g are the average ± standard deviation. n=80 cells for each circRNA. (b) Representative FISH images for circRNA (red)/ U1 snRNA(green) /parent genomic loci (purple). The parental genomic loci are indicated with white arrowheads. For each circRNA, the upper panels provide examples in which the parental genomic loci are not localised to the circRNA (red)/ U1 snRNA(green) enriched region. The lower panels demonstrate examples in which parental genomic loci are localised to circRNA (red)/ U1 snRNA (green) enriched regions (same situation as shown in Fig. 4f). In ˜65.4% (for EIF3J) and ˜51.3% of cells (for PAIP2), the two parent gene loci are localised to regions concentrated with EIciRNA and U1 snRNA (n=52 cells for EIF3J, 39 cells for PAIP2). FISH images capture the RNA locations and genomic loci at a particular moment, and we speculate that the localisation of the genomic loci and/or RNA in the nucleus may be dynamic. FISH signals for circRNA were detected with a junction probe.

Supplementary Figure 8 ChIP assay results.

(a) Flow chart of the experimental setup for pol II, U1A, and U1C ChIP after siRNA knockdown. (b) Semi-quantitative PCR corresponding to the real-time PCR results shown in Fig. 6e. (c) Association between pol II (pol II ChIP) and the gene body decreased upon siRNA-mediated knockdown of circRNA. Real-time PCR results are shown, and the sites examined within the gene body are the same as those shown in Supplementary Figure. 6b. All data are from triplicate experiments. Error bars represent the s.e.m. *, P value < 0.05, and **, P value < 0.01. P values were determined with a two-tailed Student’s t-test. (d) siRNA-mediated knockdown of circEIF3J had no effect on the association between pol II and the PAIP2 gene body or vice versa. (e) Binding of pol II to parental gene promoters was not altered by knockdown of parental mRNA. The sites examined within the gene promoter are the same as those shown in Fig. 5d.

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Supplementary Figures 1–8 and Supplementary Tables 1–4 (PDF 8362 kb)

Supplementary Data Set 1

Uncropped images corresponding to Figs. 1e and 5b (PDF 10534 kb)

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Li, Z., Huang, C., Bao, C. et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol 22, 256–264 (2015). https://doi.org/10.1038/nsmb.2959

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