Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A novel class of microRNA-recognition elements that function only within open reading frames

Nature Structural & Molecular Biology volume 25, pages 1019–1027 (2018)Cite this article

Subjects

Abstract

MicroRNAs (miRNAs) are well known to target 3′ untranslated regions (3′ UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these ‘unusual’ MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3′ side rather than the 5′ seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3′ UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: A miR-20a-target site in DAPK3 functions exclusively in CDS.
Fig. 2: CDS- and 3′-UTR-targeted miRNAs require distinct base-pairing at the 3′ end and 5′ seed.
Fig. 3: CDS-targeted miRNAs function in an Ago-dependent but GW182-independent manner.
Fig. 4: Inefficient recruitment of GW182 to CDS RISC.
Fig. 5: CDS-targeted miRNA induces aborted translation.
Fig. 6: Effects of CDS-targeted miRNAs on ribosome binding.

Similar content being viewed by others

Data availability

All deep-sequencing data generated in this study have been deposited in the Gene Expression Omnibus database under accession number GSE115146. Source data for the graphs in all figures are available online. Other data are available from the corresponding author upon reasonable request.

References

  1. Pasquinelli, A. E. MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat. Rev. Genet. 13, 271–282 (2012).

    Article  CAS  Google Scholar 

  2. Jonas, S. & Izaurralde, E. Towards a molecular understanding of microRNA-mediated gene silencing. Nat. Rev. Genet. 16, 421–433 (2015).

    Article  CAS  Google Scholar 

  3. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009).

    Article  CAS  Google Scholar 

  4. Wang, Y. et al. A new transmyocardial degradable stent combined with growth factor, heparin, and stem cells in acute myocardial infarction. Cardiovasc. Res. 84, 461–469 (2009).

    Article  CAS  Google Scholar 

  5. Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).

    Article  CAS  Google Scholar 

  6. Rajewsky, N. microRNA target predictions in animals. Nat. Genet. 38, S8–S13 (2006).

    Article  CAS  Google Scholar 

  7. Lal, A. et al. miR-24 inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to “seedless” 3’UTR microRNA recognition elements. Mol. Cell 35, 610–625 (2009).

    Article  CAS  Google Scholar 

  8. Helwak, A., Kudla, G., Dudnakova, T. & Tollervey, D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153, 654–665 (2013).

    Article  CAS  Google Scholar 

  9. van Kouwenhove, M., Kedde, M. & Agami, R. MicroRNA regulation by RNA-binding proteins and its implications for cancer. Nat. Rev. Cancer 11, 644–656 (2011).

    Article  Google Scholar 

  10. Gu, S., Jin, L., Zhang, F., Sarnow, P. & Kay, M. A. Biological basis for restriction of microRNA targets to the 3′ untranslated region in mammalian mRNAs. Nat. Struct. Mol. Biol. 16, 144–150 (2009).

    Article  CAS  Google Scholar 

  11. Duursma, A. M., Kedde, M., Schrier, M., le Sage, C. & Agami, R. miR-148 targets human DNMT3b protein coding region. RNA 14, 872–877 (2008).

    Article  CAS  Google Scholar 

  12. Tay, Y., Zhang, J., Thomson, A. M., Lim, B. & Rigoutsos, I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455, 1124–1128 (2008).

    Article  CAS  Google Scholar 

  13. Hausser, J., Syed, A. P., Bilen, B. & Zavolan, M. Analysis of CDS-located miRNA target sites suggests that they can effectively inhibit translation. Genome Res. 23, 604–615 (2013).

    Article  CAS  Google Scholar 

  14. Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96 (2013).

    Article  CAS  Google Scholar 

  15. Chi, S. W., Zang, J. B., Mele, A. & Darnell, R. B. Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature 460, 479–486 (2009).

    Article  CAS  Google Scholar 

  16. Filipowicz, W., Bhattacharyya, S. N. & Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102–114 (2008).

    Article  CAS  Google Scholar 

  17. Wu, L., Fan, J. & Belasco, J. G. MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl Acad. Sci. USA 103, 4034–4039 (2006).

    Article  CAS  Google Scholar 

  18. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005).

    Article  CAS  Google Scholar 

  19. Giraldez, A. J. et al. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79 (2006).

    Article  CAS  Google Scholar 

  20. Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).

    Article  CAS  Google Scholar 

  21. Djuranovic, S., Nahvi, A. & Green, R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science 336, 237–240 (2012).

    Article  CAS  Google Scholar 

  22. Huntzinger, E. & Izaurralde, E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat. Rev. Genet. 12, 99–110 (2011).

    Article  CAS  Google Scholar 

  23. Pillai, R. S. et al. Inhibition of translational initiation by Let-7 microRNA in human cells. Science 309, 1573–1576 (2005).

    Article  CAS  Google Scholar 

  24. Humphreys, D. T., Westman, B. J., Martin, D. I. K. & Preiss, T. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc. Natl Acad. Sci. USA 102, 16961–16966 (2005).

    Article  CAS  Google Scholar 

  25. Seggerson, K., Tang, L. & Moss, E. G. Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. Dev. Biol. 243, 215–225 (2002).

    Article  CAS  Google Scholar 

  26. Petersen, C. P., Bordeleau, M. E., Pelletier, J. & Sharp, P. A. Short RNAs repress translation after initiation in mammalian cells. Mol. Cell 21, 533–542 (2006).

    Article  CAS  Google Scholar 

  27. Nottrott, S., Simard, M. J. & Richter, J. D. Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nat. Struct. Mol. Biol. 13, 1108–1114 (2006).

    Article  CAS  Google Scholar 

  28. Maroney, P. A., Yu, Y. & Nilsen, T. W. MicroRNAs, mRNAs, and translation. Cold Spring Harb. Symp. Quant. Biol. 71, 531–535 (2006).

    Article  CAS  Google Scholar 

  29. Tat, T. T., Maroney, P. A., Chamnongpol, S., Coller, J. & Nilsen, T. W. Cotranslational microRNA mediated messenger RNA destabilization. eLife 5, e12880 (2016).

    Article  Google Scholar 

  30. Cai, Z. et al. Oncogenic miR-17/20a forms a positive feed-forward loop with the p53 kinase DAPK3 to promote tumorigenesis. J. Biol. Chem. 290, 19967–19975 (2015).

    Article  CAS  Google Scholar 

  31. O’Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V. & Mendell, J. T. c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839–843 (2005).

    Article  Google Scholar 

  32. Forman, J. J., Legesse-Miller, A. & Coller, H. A. A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence. Proc. Natl Acad. Sci. USA 105, 14879–14884 (2008).

    Article  CAS  Google Scholar 

  33. Elcheva, I., Goswami, S., Noubissi, F. K. & Spiegelman, V. S. CRD-BP protects the coding region of betaTrCP1 mRNA from miR-183-mediated degradation. Mol. Cell 35, 240–246 (2009).

    Article  CAS  Google Scholar 

  34. Takagi, S. et al. MicroRNAs regulate human hepatocyte nuclear factor 4α, modulating the expression of metabolic enzymes and cell cycle. J. Biol. Chem. 285, 4415–4422 (2010).

    Article  CAS  Google Scholar 

  35. Broughton, J. P., Lovci, M. T., Huang, J. L., Yeo, G. W. & Pasquinelli, A. E. Pairing beyond the seed supports microRNA targeting specificity. Mol. Cell 64, 320–333 (2016).

    Article  CAS  Google Scholar 

  36. Eichhorn, S. W. et al. mRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues. Mol. Cell 56, 104–115 (2014).

    Article  CAS  Google Scholar 

  37. Behm-Ansmant, I. et al. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev. 20, 1885–1898 (2006).

    Article  CAS  Google Scholar 

  38. Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference. Nat. Cell Biol. 7, 1267–1274 (2005).

    Article  Google Scholar 

  39. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    Article  CAS  Google Scholar 

  40. Liu, J. et al. A role for the P-body component GW182 in microRNA function. Nat. Cell Biol. 7, 1261–1266 (2005).

    Article  Google Scholar 

  41. Zhao, Y. et al. MicroRNA-mediated repression of nonsense mRNAs. eLife 3, e03032 (2014).

    Article  Google Scholar 

  42. Meijer, H. A. et al. Translational repression and eIF4A2 activity are critical for microRNA-mediated gene regulation. Science 340, 82–85 (2013).

    Article  CAS  Google Scholar 

  43. Donnelly, M. L. et al. The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring `2A-like' sequences. J. Gen. Virol. 82, 1027–1041 (2001).

    Article  CAS  Google Scholar 

  44. Kim, J. H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, e18556 (2011).

    Article  CAS  Google Scholar 

  45. Ishimura, R. et al. Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration. Science 345, 455–459 (2014).

    Article  CAS  Google Scholar 

  46. Buskirk, A. R. & Green, R. Ribosome pausing, arrest and rescue in bacteria and eukaryotes. Phil. Trans. R. Soc. Lond. B 372, 20160183 (2017).

    Article  Google Scholar 

  47. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

    Article  CAS  Google Scholar 

  48. Grosswendt, S. et al. Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol. Cell 54, 1042–1054 (2014).

    Article  CAS  Google Scholar 

  49. Ishigaki, Y., Li, X., Serin, G. & Maquat, L. E. Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607–617 (2001).

    Article  CAS  Google Scholar 

  50. Doma, M. K. & Parker, R. Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440, 561–564 (2006).

    Article  CAS  Google Scholar 

  51. Shoemaker, C. J., Eyler, D. E. & Green, R. Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science 330, 369–372 (2010).

    Article  CAS  Google Scholar 

  52. Presnyak, V. et al. Codon optimality is a major determinant of mRNA stability. Cell 160, 1111–1124 (2015).

    Article  CAS  Google Scholar 

  53. Iwakawa, H. O. & Tomari, Y. Molecular insights into microRNA-mediated translational repression in plants. Mol. Cell 52, 591–601 (2013).

    Article  CAS  Google Scholar 

  54. Woolstenhulme, C. J., Guydosh, N. R., Green, R. & Buskirk, A. R. High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep. 11, 13–21 (2015).

    Article  CAS  Google Scholar 

  55. Bhattacharyya, S. N., Habermacher, R., Martine, U., Closs, E. I. & Filipowicz, W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125, 1111–1124 (2006).

    Article  CAS  Google Scholar 

  56. Parker, R. & Sheth, U. P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

    Article  CAS  Google Scholar 

  57. Decker, C. J. & Parker, R. P-bodies and stress granules: possible roles in the control of translation and mRNA degradation. Cold Spring Harb. Perspect. Biol. 4, a012286 (2012).

    Article  Google Scholar 

  58. Aizer, A. et al. Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage. J. Cell Sci. 127, 4443–4456 (2014).

    Article  CAS  Google Scholar 

  59. Ishimura, R., Nagy, G., Dotu, I., Chuang, J. H. & Ackerman, S. L. Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation. eLife 5, e14295 (2016).

    Article  Google Scholar 

  60. Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508–514 (2016).

    Article  Google Scholar 

  61. Adiconis, X. et al. Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat. Methods 10, 623–629 (2013).

    Article  CAS  Google Scholar 

  62. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  Google Scholar 

  63. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013)..

    Article  Google Scholar 

  64. Loeb, G. B. et al. Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting. Mol. Cell 48, 760–770 (2012).

    Article  CAS  Google Scholar 

  65. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  Google Scholar 

  66. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  Google Scholar 

  67. Rehmsmeier, M., Steffen, P., Hochsmann, M. & Giegerich, R. Fast and effective prediction of microRNA/target duplexes. RNA 10, 1507–1517 (2004).

    Article  CAS  Google Scholar 

  68. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Ministry of Science and Technology of China (2017YFA0506400, Y. Zhou and X.-D.F.), the National Natural Science Foundation of China (31670827 and 91640115, Y. Zhou), the Chinese 111 program grant (B06018, X.-D.F.), Chinese Academy of Science foundation grant (22KJZD-EW-L12, X.Z.) and NIH grants (HG004659, GM049369 and GM052872, X.-D.F.). pRL-SV40 plasmids carrying IRESs from EMCV, HCV, and CrPV were generous gifts from M. Bushell (Medical Research Council Toxicology Unit, UK).

Author information

Author notes

  1. These authors contributed equally: Kai Zhang, Xiaorong Zhang, Zhiqiang Cai, Jie Zhou.

Authors and Affiliations

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

    Kai Zhang, Zhiqiang Cai, Jie Zhou, Ran Cao, Zonggui Chen, Dehe Wang, Wen Ruan, Yu Zhou & Xiang-Dong Fu

  2. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Xiaorong Zhang, Qian Zhao, Guangqiao Liu, Yuanchao Xue, Yan Qin & Xiang-Dong Fu

  3. Jiangsu Key Laboratory of Experimental & Translational Non-coding RNA Research, Institute of Translational Medicine, School of Medicine, Yangzhou University, Yangzhou, China

    Ya Zhao

  4. Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, China

    Ya Zhao & Ligang Wu

  5. Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA, USA

    Bing Zhou & Xiang-Dong Fu

  6. Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, OH, USA

    Timothy Nilsen

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

    Yu Zhou

Authors
  1. Kai Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaorong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhiqiang Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jie Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ran Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ya Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zonggui Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dehe Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wen Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Qian Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Guangqiao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yuanchao Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yan Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Bing Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Ligang Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Timothy Nilsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Yu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Xiang-Dong Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z. Cai, K.Z., X.Z., Y. Zhou, T.N., and X.-D.F. designed the experiments; Z. Cai, K.Z., X.Z., and W.R. performed most experiments; J.Z., B.Z., Z. Chen, D.W., and Y. Zhou analyzed the data; Y. Zhao and L.W. performed the deadenylation assay; R.C., G.L., Q.Z., Y.X., and Y.Q. contributed additional data. Z. Cai, K.Z., Y. Zhou, T.N., and X.-D.F. wrote the paper.

Corresponding authors

Correspondence to Yu Zhou or Xiang-Dong Fu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 MicroRNA represses target proteins without causing mRNA decay.

a,b, Western blotting analysis of miR-20a target genes in response to increasing doses of miR-20a mimic (a) or antagomir (b). c,d, RT-qPCR analysis of miR-20a levels in HeLa cells transfected with miR-20a mimic (c) or antagomir (d). e,f, RT-qPCR analysis of DAPK3 mRNA levels in HeLa cells transfected with miR-20a mimic (e) or antagomir (f). g, Luciferase reporter constructs containing the miR-20a MRE from DAPK3 (native) in Renilla 5’ UTR or mutated to restore base-pairing in the 5’ end (seed) coupled with progressive mismatches (3MM+seed) in the 3’ end. h,i, Results of the luciferase reporter assays (h) and quantification of luciferase mRNA (i). j,k, mRNA levels of luciferase reporters containing individual MREs from 4 indicated genes that function in both CDS and 3’UTR (j) or those that function only in CDS (k), as shown in main Fig. 2e,f. l,m, Luciferase activities from the reporters containing individual MREs from 4 indicated genes that function only in CDS in response to co-transfected Let-7b mimic (l) or antagomir (m). NC: Non-specific oligo control; SC: Scrambled RNA oligo control. n, Western blotting analysis of three additional Let-7b target genes. The three gene products were analyzed in HeLa cells transfected with non-specific RNA oligonucleotide control (NC), Let-7b mimic, or antagomir, as indicated. Actin served as loading control. The data in c,d,e,f,h,i,j,k,l,m were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).

Source data

Supplementary Figure 2 Mutation analysis of multiple Let-7b MREs in CDS regions of individual genes.

a,b,c,d, Deduced base-paring between Let-7b and its targets in individual genes that function only in CDS, as described in main Fig. 2f. A panel of mutants in each targeting site is also shown with calculated base-pairing potential (∆G). The constructs on the left of each panel contain progressive mutations in 3’ side and the constructs on the right of each panel carry progressive mutations in 3’ side with restored base-pairing in 5’ seed (red-labeled nucleotides). e,f, Luciferase activities of individual reporters containing different mutations, without (e) or with restored seed (f), as indicated. The data in e and f were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).

Source data

Supplementary Figure 3 Luciferase activities and mRNA levels of the reporters with or without restored seed.

a, Constructs containing various MREs after restoring seed base pairing (right) inserted in 3’UTR. b,c, Luciferase activities (b) and mRNA levels (c) in HeLa cells transfected with seed restored constructs in a. d, Constructs containing various ERAP2-derived MREs with decreasing base-pairing potentials (∆G) in 5’ seed inserted in CDS. e, Luciferase activities (upper panel) and mRNA levels (lower panel) in HeLa cells transfected with the constructs in d. The data in b,c,e were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).

Source data

Supplementary Figure 4 Functional test for Ago2 or GW182 dependency and for CDS-targeted miRNA to induce translation abortion.

a, One miR-20a MRE and four Let-7b MREs were tested in wild-type or Ago2 knockout MEFs. Note that Ago2 knockout MEFs contain a small population of undeleted cells, which likely contributed to the basal level of Ago2, as shown by western blotting on top. The MREs inserted in both CDS (black bar) or 3’UTR (grey bar) were tested. b, One miR-20a MRE and four Let-7b MREs were tested in non-specific siRNA-treated (NC) or siGW182-treated HeLa cells. Knockdown efficiency was determined by western blotting, as shown on top. The MREs inserted in both CDS (black bar) or 3’UTR (gray bar) were tested. The data shown in a and b were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). c, Western blotting analysis of endogenous proteins in non-specific siRNA-treated (-) or siGW182-treated (+) HeLa in response to miR-20a mimic (left) or antagomir (right). d, Repeated experiments as in main Fig. 4b. Western blotting analysis of Ago2 and GW182 associated with the Renilla luciferase reporter carrying perfect seed base-pairing (“seed”) or imperfect seed with or without additional 3nt mismatches in 3’ end (“3p3MM+seed” and “3p3MM”) in CDS or 3’UTR. Equal amounts of captured reporter mRNAs were detected by RT-PCR and the Firfly reporter served as a negative control. e, Repeated experiments as in main Fig. 5b. Western blotting of the FLAG-tagged luciferase reporter proteins expressed in transfected HeLa cells. The second to last lane was from cells co-transfected with the reporter containing the miR-20a MRE and the miR-20a antagomir. f, Repeated experiments as in main Fig. 5c. Western blotting of GFP-2A-MRE-2A-mCherry reporter proteins expressed in transfected HeLa cells. Co-transfected FLAG-tagged Renilla served as a loading control.

Source data

Supplementary Figure 5 Genome-wide analysis of Ago2 eCLIP, ribo-seq, and RNA-seq data.

a, The Pearson coefficient between two biological replicates of Ago2 eCLIP-seq on HeLa cells before (left) and after miR-20a overexpression (right). b, Percentage of sequences underlying Ago2 eCLIP peaks in 3’UTR that can base pair with miRNA seed. Left, Enriched Ago2 peaks in 3’UTR for top 20 mostly expressed miRNAs in comparison with non-enriched and random sites. Right, miR-20a induced Ago2 peaks in 3’UTR compared to uninduced and random sites. c, Meta-profiles of Ago2 binding on miR-20a induced (left) and uninduced (right) sites. d, Boxplots of ΔG between miR-20a and sequences underlying miR-20a induced Ago2 peaks (red) in CDS and 3’UTR in comparison with uninduced (blue) and random (gray) sites. Data from two independent eCLIP replicates are separately displayed. The median value for each group is shown with a horizontal gray line. Filled boxes extend from the first to the third quartile. The upper/lower whisker extends from the hinge to the highest/lowest value that is within 1.5 * IQR of the hinge, where IQR is the inter-quartile range. Data beyond the end of the whiskers are outliers and plotted as points. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). e, The Pearson coefficient among three Ribo-seq and two RNA-seq experiments under before (WT) and after miR-20a overexpression (OE). f, Length distribution of ribosome protected fragments (RPFs) from three repeats under WT and OE conditions. g, The 3nt periodicity of RPFs with different lengths. h,i, Meta-gene analysis of RPFs on miR-20a induced Ago2 peaks (left) in comparison with uninduced Ago2 peaks (middle) and random sites (right). Data from two independent Ago2 eCLIP experiments (h, Repeat 1; i, Repeat 2) were used to perform the meta-gene analysis. See the data with combined data in main Fig. 6d.

Supplementary Figure 6 CDS-targeted miRNA does not alter overall polysome profiles.

a, Luciferase activities (upper panel) and relative mRNA levels determined by real-time RT-PCR (lower panel) in three stable HeLa cell lines expressing the luciferase reporter carrying the miR-20a MRE in different locations (note that, as shown in Fig. 2a, the native MRE is non-functional in 3’UTR, which served as control). b,c,d, Polysome profiling by sucrose gradients on the three stable HeLa cell lines. OD260 traces are shown below individual constructs and the distribution of mRNAs from individual reporters and the endogenous Actin gene is shown at bottom. The mRNA levels were determined by real-time RT-PCR on individual fractions and expressed as percentage of the sum. The data shown in a,b,c,d were based on 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test).

Source data

Supplementary Information

Supplementary Figures 1–6 and Supplementary Table 1

Supplementary Figures 1–6 and Supplementary Table 1

Reporting Summary

Supplementary Data Set 1

Western blotting and original gel images

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, K., Zhang, X., Cai, Z. et al. A novel class of microRNA-recognition elements that function only within open reading frames. Nat Struct Mol Biol 25, 1019–1027 (2018). https://doi.org/10.1038/s41594-018-0136-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-018-0136-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing