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mRNA structural dynamics shape Argonaute-target interactions

Nature Structural & Molecular Biology volume 27, pages 790–801 (2020)Cite this article

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An Author Correction to this article was published on 04 June 2021

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Abstract

Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by guiding Argonaute2 (AGO2) to its target site. Target site accessibility is critical for AGO2-target interactions, but how target site accessibility is controlled in vivo is poorly understood. Here, we use live-cell single-molecule imaging in human cells to determine rate constants of the AGO2 cleavage cycle in vivo. We find that the rate-limiting step in mRNA cleavage frequently involves unmasking of target sites by translating ribosomes. Target site masking is caused by heterogeneous intramolecular RNA-RNA interactions, which can conceal target sites for many minutes in the absence of translation. Our results uncover how dynamic changes in mRNA structure shape AGO2-target recognition, provide estimates of mRNA folding and unfolding rates in vivo, and provide experimental evidence for the role of mRNA structural dynamics in control of mRNA-protein interactions.

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Fig. 1: Observing AGO2-dependent mRNA target silencing by single-molecule live-cell imaging.
Fig. 2: Ribosomes stimulate AGO2-dependent mRNA cleavage by promoting AGO2-target interactions.
Fig. 3: In vivo kinetics of the AGO2 cleavage cycle.
Fig. 4: Masking of mRNA target sites by RNA structures inhibits AGO2-target interactions.
Fig. 5: Multiple weak intramolecular mRNA interactions cooperatively mask AGO2 target sites.
Fig. 6: Kinetics of mRNA folding shape AGO2-mRNA interactions.

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Data availability

A selection of the raw imaging data (related to Figs. 1–6) used in this study is available on Mendeley (https://doi.org/10.17632/h2r32zhgwn.1). Source data are available with the paper online.

Code availability

Custom code used in this study is available on Mendeley (https://doi.org/10.17632/h2r32zhgwn.1). Source data are provided with this paper.

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References

  1. Bartel, D. P. Metazoan microRNAs. Cell 173, 20–51 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Gebert, L. F. R. & MacRae, I. J. Regulation of microRNA function in animals. Nat. Rev. Mol. Cell Biol. 20, 21–37 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94–108 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656–668 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ozata, D. M., Gainetdinov, I., Zoch, A., O’Carroll, D. & Zamore, P. D. PIWI-interacting RNAs: small RNAs with big functions. Nat. Rev. Genet. 20, 89–108 (2019).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  7. Iwasaki, Y. W., Siomi, M. C. & Siomi, H. PIWI-interacting RNA: its biogenesis and functions. Annu. Rev. Biochem. 84, 405–433 (2015).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  9. Meister, G. Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–459 (2013).

    CAS  PubMed  Google Scholar 

  10. Schirle, N. T., Sheu-Gruttadauria, J. & MacRae, I. J. Structural basis for microRNA targeting. Science 346, 608–613 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    CAS  PubMed  Google Scholar 

  12. Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C. A dynamic search process underlies microRNA targeting. Cell 162, 96–107 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Jo, M. H. et al. Human Argonaute 2 has diverse reaction pathways on target RNAs. Mol. Cell 59, 117–124 (2015).

    CAS  PubMed  Google Scholar 

  14. Salomon, W. E., Jolly, S. M., Moore, M. J., Zamore, P. D. & Serebrov, V. Single-molecule imaging reveals that argonaute reshapes the binding properties of its nucleic acid guides. Cell 162, 84–95 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Wee, L. M., Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151, 1055–1067 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Yao, C., Sasaki, H. M., Ueda, T., Tomari, Y. & Tadakuma, H. Single-molecule analysis of the target cleavage reaction by the drosophila RNAi enzyme complex. Mol. Cell 59, 125–132 (2015).

    CAS  PubMed  Google Scholar 

  17. Jung, S. R. et al. Dynamic anchoring of the 3′-end of the guide strand controls the target dissociation of Argonaute-guide complex. J. Am. Chem. Soc. 135, 16865–16871 (2013).

    CAS  PubMed  Google Scholar 

  18. Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    CAS  PubMed  Google Scholar 

  19. Sheu-Gruttadauria, J. & MacRae, I. J. Structural foundations of RNA silencing by Argonaute. J. Mol. Biol. 429, 2619–2639 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327–341 (2018).

    CAS  PubMed  Google Scholar 

  21. Jankowsky, E. & Harris, M. E. Specificity and nonspecificity in RNA-protein interactions. Nat. Rev. Mol. Cell Biol. 16, 533–544 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    CAS  PubMed  Google Scholar 

  23. Kedde, M. et al. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell 131, 1273–1286 (2007).

    CAS  PubMed  Google Scholar 

  24. Grimson, A. et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91–105 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ameres, S. L., Martinez, J. & Schroeder, R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130, 101–112 (2007).

    CAS  PubMed  Google Scholar 

  27. Beaudoin, J. D. et al. Analyses of mRNA structure dynamics identify embryonic gene regulatory programs. Nat. Struct. Mol. Biol. 25, 677–686 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Becker, W. R. et al. High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2. Mol. Cell 75, 741–755.e11 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Brown, K. M., Chu, C. Y. & Rana, T. M. Target accessibility dictates the potency of human RISC. Nat. Struct. Mol. Biol. 12, 469–470 (2005).

    CAS  PubMed  Google Scholar 

  30. Kertesz, M., Iovino, N., Unnerstall, U., Gaul, U. & Segal, E. The role of site accessibility in microRNA target recognition. Nat. Genet. 39, 1278–1284 (2007).

    CAS  PubMed  Google Scholar 

  31. Tafer, H. et al. The impact of target site accessibility on the design of effective siRNAs. Nat. Biotechnol. 26, 578–583 (2008).

    CAS  PubMed  Google Scholar 

  32. Chen, S. J. RNA folding: conformational statistics, folding kinetics, and ion electrostatics. Annu. Rev. Biophys. 37, 197–214 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Ganser, L. R., Kelly, M. L., Herschlag, D. & Al-Hashimi, H. M. The roles of structural dynamics in the cellular functions of RNAs. Nat. Rev. Mol. Cell Biol. 20, 474–489 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Solomatin, S. V., Greenfeld, M., Chu, S. & Herschlag, D. Multiple native states reveal persistent ruggedness of an RNA folding landscape. Nature 463, 681–684 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Ditzler, M. A., Rueda, D., Mo, J., Hakansson, K. & Walter, N. G. A rugged free energy landscape separates multiple functional RNA folds throughout denaturation. Nucleic Acids Res. 36, 7088–7099 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Pichon, X. et al. Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells. J. Cell Biol. 214, 769–781 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yan, X., Hoek, T. A., Vale, R. D. & Tanenbaum, M. E. Dynamics of translation of single mRNA molecules in vivo. Cell 165, 976–989 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science 352, 1425–1429 (2016).

    CAS  PubMed  Google Scholar 

  39. Wu, B., Eliscovich, C., Yoon, Y. J. & Singer, R. H. Translation dynamics of single mRNAs in live cells and neurons. Science 352, 1430–1435 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Wang, C., Han, B., Zhou, R. & Zhuang, X. Real-time imaging of translation on single mRNA transcripts in live cells. Cell 165, 990–1001 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Horvathova, I. et al. The dynamics of mRNA turnover revealed by single-molecule imaging in single cells. Mol. Cell 68, 615–625.e9 (2017).

    CAS  PubMed  Google Scholar 

  42. Lam, J. K., Chow, M. Y., Zhang, Y. & Leung, S. W. siRNA versus miRNA as therapeutics for gene silencing. Mol. Ther. Nucleic Acids 4, e252 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Hoek, T. A. et al. Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay. Mol. Cell 75, 324–339.e11 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Tanenbaum, M. E., Vale, R. D., Stern-Ginossar, N. & Weissman, J. S. Regulation of mRNA translation during mitosis. Elife 4, e07957 (2015).

    PubMed Central  Google Scholar 

  45. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lu, Z. et al. RNA duplex map in living cells reveals higher-order transcriptome structure. Cell 165, 1267–1279 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Metkar, M. et al. Higher-Order Organization Principles of Pre-translational mRNPs. Mol. Cell 72, 715–726.e3 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Strobel, E. J., Yu, A. M. & Lucks, J. B. High-throughput determination of RNA structures. Nat. Rev. Genet. 19, 615–634 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Gong, J. et al. RISE: a database of RNA interactome from sequencing experiments. Nucleic Acids Res. 46, D194–D201 (2018).

    CAS  PubMed  Google Scholar 

  50. Aw, J. G. et al. In vivo mapping of eukaryotic RNA interactomes reveals principles of higher-order organization and regulation. Mol. Cell 62, 603–617 (2016).

    CAS  PubMed  Google Scholar 

  51. Rouskin, S., Zubradt, M., Washietl, S., Kellis, M. & Weissman, J. S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705 (2014).

    CAS  PubMed  Google Scholar 

  52. Wan, Y. et al. Landscape and variation of RNA secondary structure across the human transcriptome. Nature 505, 706–709 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat. Methods 14, 75–82 (2017).

    CAS  PubMed  Google Scholar 

  54. Mustoe, A. M. et al. Pervasive regulatory functions of mRNA structure revealed by high-resolution SHAPE probing. Cell 173, 181–195.e18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Bevilacqua, P. C., Ritchey, L. E., Su, Z. & Assmann, S. M. Genome-wide analysis of RNA secondary structure. Annu. Rev. Genet. 50, 235–266 (2016).

    CAS  PubMed  Google Scholar 

  56. Ding, Y. et al. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505, 696–700 (2014).

    CAS  PubMed  Google Scholar 

  57. Adivarahan, S. et al. Spatial organization of single mRNPs at different stages of the gene expression pathway. Mol. Cell 72, 727–738.e5 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Mizrahi, O. et al. Virus-Induced Changes in mRNA Secondary Structure Uncover cis-Regulatory Elements that Directly Control Gene Expression. Mol. Cell 72, 862–874.e5 (2018).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Tauber, D. et al. Modulation of RNA condensation by the DEAD-box protein eIF4A. Cell 180, 411–426.e16 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Golden, R. J. et al. An Argonaute phosphorylation cycle promotes microRNA-mediated silencing. Nature 542, 197–202 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Lyubimova, A. et al. Single-molecule mRNA detection and counting in mammalian tissue. Nat. Protoc. 8, 1743–1758 (2013).

    PubMed  Google Scholar 

  64. Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465, 818–822 (2010).

    CAS  PubMed  Google Scholar 

  65. Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates. RNA 19, 271–279 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Joo, C. & Ha, T. Single-molecule FRET with total internal reflection microscopy. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.top072058 (2012).

  67. Chandradoss, S. D. et al. Surface passivation for single-molecule protein studies. J. Vis. Exp. 86, e50549 (2014).

    Google Scholar 

  68. Pan, H., Xia, Y., Qin, M., Cao, Y. & Wang, W. A simple procedure to improve the surface passivation for single molecule fluorescence studies. Phys. Biol. 12, 045006 (2015).

    PubMed  Google Scholar 

  69. Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using microManager. Curr. Protoc. Mol. Biol. 92, 14.20.1–14.20.17 (2010).

    Google Scholar 

  70. Ruijtenberg, S., Hoek, T. A., Yan, X. & Tanenbaum, M. E. Imaging translation dynamics of single mRNA molecules in live cells. Methods Mol. Biol. 1649, 385–404 (2018).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Depken for helpful discussions with the computational modeling. We thank L. Steller, I. Bally, and R. Banerjee for help with experiments. We would also like to thank the Tanenbaum lab members for helpful discussions and T. Hoek and D. Khuperkar for critical reading of the manuscript. This work was financially supported by the European Research Council (ERC) through an ERC starting grant (ERCSTG 677936-RNAREG) to M.E.T., a VENI grant from the Netherlands Organization for Scientific Research (NWO) (NWO 016.VENI.171.050) to S.R., an ERC consolidator grant (819299) and a VIDI grant from NWO (864.14.002) to C.J., and the National Institute of General Medical Sciences (R35 GM127090) to I.J.M.; M.E.T., S.R., S.S., D.d.S. and I.L. are supported by the Oncode Institute that is partly funded by the Dutch Cancer Society (KWF).

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  1. Suzan Ruijtenberg

    Present address: Developmental Biology, Department of Biology, Faculty of Sciences, Utrecht University, Utrecht, the Netherlands

  2. These authors contributed equally: Suzan Ruijtenberg, Stijn Sonneveld.

Authors and Affiliations

  1. Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, the Netherlands

    Suzan Ruijtenberg, Stijn Sonneveld, Ive Logister, Dion de Steenwinkel & Marvin E. Tanenbaum

  2. Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, Delft, the Netherlands

    Tao Ju Cui & Chirlmin Joo

  3. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Yao Xiao & Ian J. MacRae

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Contributions

S.R., S.S. and M.E.T. conceived the project; S.R., S.S., I.L., and D.d.S. performed the in vivo experiments and analyzed the data; S.S. performed the computational modeling; T.J.C. performed the in vitro experiments and analyzed the data under supervision of C.J.; Y.X. purified the hAGO2 complex under supervision of I.J.M.; S.R., S.S. and T.J.C. prepared the figures; S.R., S.S. and M.E.T. wrote the manuscript; and T.J.C., Y.X., I.J.M. and C.J. provided input.

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Correspondence to Marvin E. Tanenbaum.

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Extended data

Extended Data Fig. 1 Effects of AGO2-siRNA complexes on mRNA transcription and translation.

a, Relative mRNA levels of endogenous KIF18B based on qPCR in non-transfected cells (no siRNA) and cells transfected with KIF18B siRNA #1 (+ siRNA). Each dot represents an independent experiment and lines with error bars indicate the mean ± SEM. b-f, i, Cells expressing the KIF18B reporter without siRNA (no siRNA) or transfected with 10 nM KIF18B siRNA #1 (+ siRNA) were fixed and incubated with smFish probes to visualize reporter mRNAs. b, Representative images of cells incubated with smFISH probes targeting the KIF18B reporter (SunTag-Cy5 and PP7-Alexa594) in no siRNA cells (upper panel) and + siRNA cells (lower panel). Arrows in insets indicate mRNA molecules for which the 5′ end (SunTag-Cy5 probe) and 3′ end (PP7-Alexa594 probe) do not co-localize. Scale bar, 10 µm in large images and 1 µm in insets. c-d, Number of mRNAs in no siRNA and + siRNA cells in (c) the cytoplasm and (d) the nucleus determined based on smFISH using probes targeting the SunTag sequence. Each dot represents a single cell and lines with error bars indicate the mean ± SEM. e-f, Percentage of mRNAs for which the 5′ end (labeled with SunTag probes) and 3′ end (labeled with PP7 probes) co-localized in no siRNA and + siRNA cells, either in (e) the cytoplasm or (f) the nucleus. Each dot represents a single cell and lines with error bars indicate the mean ± SEM. g, Relative AGO2 mRNA levels based on qPCR in control cells (no gRNA) and cells treated with a CRISPRi guide targeting endogenous AGO2 (AGO2 gRNA). Each dot represents an independent experiment and lines with error bars indicate the mean ± SEM. h, SunTag-PP7 cells expressing the KIF18B reporter were transfected with KIF18B siRNA #1. The number of ribosomes present on the 5′ cleavage fragment was determined one frame after the moment of cleavage (see Supplementary Note 4). Dotted red line indicates the intensity of a single SunTag array (that is the intensity associated with a single ribosome). i, Cells were treated for 40 min with dox and the integrated intensity of transcription sites was determined with smFISH probes targeting the SunTag sequence. Each dot represents a single transcription site and lines with error bars indicate the mean ± SEM. j-k, SunTag-PP7 cells expressing the KIF18B reporter were untransfected (no siRNA) or transfected with KIF18B siRNA #1 (+ siRNA). j, GFP intensity over time associated with individual mRNAs is shown for no siRNA cells (black line) and + siRNA cells (grey lines). Black line indicates average of all mRNAs in no siRNA cells, while each grey line represents the average GFP intensity of all mRNAs cleaved at the same moment relative to the start of translation (see Supplementary Note 5). The red dot indicates the moment of cleavage. k, Average increase in GFP fluorescence intensity either between 1.5-4 min after the start of translation (no siRNA) or at the moment preceding mRNA cleavage (+ siRNA) is shown (see Supplementary Note 5). Each dot represents the average of an independent experiment and lines with error bars indicate the mean ± SEM. a, c-f, g, i, P-values are based on a two-tailed Student’s t-test. k, P-value is based on a paired two-tailed t-test. P-values are indicated as * (p < 0.05), ** (p < 0.01), *** (p < 0.001), ns = not significant. Number of measurements for each experiment is listed in Supplementary Table 1. Data for graphs in a,c-k are available as source data.

Source data

Extended Data Fig. 2 Ribosomes stimulate AGO2-dependent mRNA cleavage.

a, The moment at which the first ribosome arrived at the stop codon was calculated for indicated reporters. The experimental data (colored bars) was fit with a gamma distribution (black lines) (See Supplementary Note 5). b-i, SunTag-PP7 cells expressing the indicated reporters were transfected with 50 nM (KIF18B siRNA #3) or 10 nM (all others) siRNA and treated with CHX, where indicated. The time from first detection of translation or from CHX addition until separation of GFP and mCherry foci (that is mRNA cleavage) is shown. Solid lines and corresponding shaded regions represent mean ± SEM. Dotted line indicates that the data is replotted from an earlier figure panel for comparison. j, Ratio of non-nuclear and nuclear mRNAs 90 min after addition of dox in cells expressing the KIF18B reporter (control) or KIF18B-early-stop reporter (Stop) as determined by smFISH using SunTag probes. Note that mRNA localization is similar for the two cell lines used for northern blot analysis (see Fig. 2e). Each dot represents one cell and lines with error bars indicate the mean ± SEM. P-value is based on a two-tailed Student’s t-test. k, SunTag-PP7 cells expressing the indicated reporters were transfected with 10 nM siRNA and treated with CHX, where indicated. The time from first detection of translation or from CHX addition (+ CHX) until separation of GFP and mCherry foci (that is mRNA cleavage) is shown. Solid lines and corresponding shaded regions represent mean ± SEM. l, The fraction of mRNAs that contains a ribosome on the 3′ cleavage fragment is shown for mRNAs on which translation started at least 7.5 minutes (KIF18B) or 6 minutes (GAPDH) before the moment of cleavage. On these mRNAs it is expected that the first ribosome has passed the AGO2 target site in ~95% of mRNAs (indicated by black bars) based on the experimentally-derived ribosome elongation rate. The expected fraction (black bars) and observed fraction (green bars) of mRNAs that contains a ribosome on the 3′ cleavage fragment is shown. Number of measurements for each experiment is listed in Supplementary Table 1. Data for graphs in a-l are available as source data.

Source data

Extended Data Fig. 3 In vivo and in vitro kinetics of the AGO2 cleavage cycle.

a, In vitro AGO2 cleavage reaction with purified AGO2 loaded with KIF18B siRNA #1 and a short oligonucleotide target containing the KIF18B siRNA #1 target sequence. b, Quantification of the cleaved fraction of blot in (a). c, Calculated cleavage rates in the presence of translating ribosomes are shown for different siRNA concentrations (see Supplementary Note 4). Dots and error bars indicate the mean ± SEM. Number of measurements for each experiment is listed in Supplementary Table 1. Data for graphs in b,c are available as source data.

Source data

Extended Data Fig. 4 Degree of structural masking depends on the AGO2 binding sequence and the surrounding sequence.

a, Schematic of the KIF18B reporter in which the position of the siRNA #1 and siRNA #2 binding sites are swapped. b-c, SunTag-PP7 cells expressing indicated reporters were transfected with (b) 10 nM KIF18B siRNA #1 or (c) 10 nM KIF18B siRNA #2. The time from first detection of translation until separation of GFP and mCherry foci (that is mRNA cleavage) is shown. Solid lines and corresponding shaded regions represent mean ± SEM. Dotted lines indicate that the data is replotted from an earlier figure panel for comparison. d, Schematic of the GAPDH reporter in which the KIF18B siRNA #1 or KIF18B siRNA #2 binding site is placed at the position of GAPDH siRNA #3. e-f, SunTag-PP7 cells expressing the indicated reporters were transfected with (e) 10 nM KIF18B siRNA #1 or (f) 10 nM KIF18B siRNA #2. The time from first detection of translation or CHX addition until mRNA cleavage is shown. Note that data of the KIF18B-early-stop reporter and KIF18B reporter treated with CHX are combined to generate the cleavage curve for cleavage in the absence of ribosomes. Solid lines and corresponding shaded regions represent mean ± SEM. g, Ratio of cleavage rate in the presence and absence of ribosomes is shown for the indicated siRNAs and reporters (see Supplementary Note 4). Each dot represents a single experiment and lines with error bars indicate the mean ± SEM. P-values are based on a two-tailed Student’s t-test. P-values are indicated as * (p < 0.05), ** (p < 0.01), *** (p < 0.001). K1, K2 and G3 indicate the position of the indicated siRNA. K1 refers to the position of KIF18B siRNA #1, K2 to KIF18B siRNA #2 and G3 to GAPDH siRNA #3. Light blue and light green data points are replotted from an earlier experiment. Number of measurements for each experiment is listed in Supplementary Table 1. Data for graphs in b,c,e-g are available as source data.

Source data

Extended Data Fig. 5 Multiple weak intramolecular mRNA interactions together result in potent AGO2 target site masking.

a-e, SunTag-PP7 cells expressing the indicated reporters were transfected with (a) 10 nM KIF18B siRNA #1 or (b-e) 10 nM GAPDH siRNA #3. The time from first detection of translation until separation of GFP and mCherry foci (that is mRNA cleavage) is shown. Solid lines and corresponding shaded regions represent mean ± SEM. Dotted lines indicate that the data is replotted from an earlier figure panel for comparison. f, Cleavage rates for the ‘luciferase’ reporters with indicated siRNA target sites and with different distances between the stop codon and the siRNA target site are shown. Each dot and error bar indicate the mean ± SEM. Dotted lines are only for visualization. Number of measurements for each experiment is listed in Supplementary Table 1. Data for graphs in a-f are available as source data.

Source data

Extended Data Fig. 6 Structural dynamics of RNA folding.

a-c, SunTag-PP7 cells expressing the indicated reporters were transfected with 10 nM of the indicated siRNA and treated with CHX, where indicated. The CHX cleavage curves (red lines) only include mRNAs for which translation started between (a) 2.5-5.0 min, (b) 2.0-4.5 min, or (c) 2.0-5.0 min before CHX addition (see Supplementary Note 4). Dotted lines represent optimal fit with a two-component exponential decay distribution. The no CHX cleavage curve is re-normalized and plotted from (a) 2.5 min or (b-c) 2.0 min after the start of translation. d, Relative GFP fluorescence intensities were measured before and after the addition of CHX in SunTag-PP7 cells expressing the KIF18B reporter. Intensity-time traces were aligned at the moment of CHX addition. GFP fluorescence intensities were normalized to the GFP fluorescence intensities at the moment of CHX addition. The thick blue line represents the average intensity of all traces, thin grey lines represent intensity traces of multiple single mRNAs. e, Fitting parameters and corresponding half-lives of the two-component exponential fits from Fig. 6a and Extended Data Fig. 6a, b. f, Average number of ribosomes per mRNA molecule for the KIF18B-uORF and KIF18B reporters. Each dot represents an independent experiment and lines with error bars indicate the mean ± SEM. g, i, SunTag-PP7 cells expressing the indicated reporter were transfected with the indicated siRNA and (i) treated with CHX. g, The time from first detection of translation until separation of GFP and mCherry foci (that is mRNA cleavage) is shown or i, the time from CHX addition until mRNA cleavage is shown. Solid lines and corresponding shaded regions represent mean ± SEM. Dotted lines indicate that the data is replotted from an earlier figure panel for comparison. h, Ratio of the cleavage rates in the presence and absence of translating ribosomes is shown for the indicated siRNAs and reporters (see Supplementary Note 4). Each dot represents a single experiment and lines with error bars indicate the mean ± SEM. Light black data points are replotted from an earlier experiment. j, Simulated cleavage curves for 10 and 0.1 nM siRNA concentration using fast or slow unmasking rates (average unmasking time of 1 s and 1,200s, respectively). Number of measurements for each experiment is listed in Supplementary Table 1. Data for graphs in a-d,f-j are available as source data.

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Supplementary Notes 1−8, Supplementary Table 1 and Supplementary References.

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Supplementary Video 1

SunTag-PP7 cells expressing the KIF18B reporter (Fig. 1b). Images were acquired every 30 s on a spinning-disk confocal microscope focusing near the bottom plasma membrane of the cell. Individual mRNAs can be tracked for the duration of the movie, undergoing many rounds of translation.

Supplementary Video 2

SunTag-PP7 cells expressing the KIF18B reporter (Fig. 1b) were transfected with 10 nM of KIF18B siRNA #1. Images were acquired every 30 s on a spinning-disk confocal microscope focusing near the bottom plasma membrane of the cell. Individual mRNAs can be tracked from the start of translation (appearance of GFP signal) until mRNA cleavage (separation of mCherry and GFP foci) or for the duration of the movie. Note that the mCherry signal disappears after cleavage, indicating exonucleolytic decay of the 3′ cleavage fragment.

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Ruijtenberg, S., Sonneveld, S., Cui, T.J. et al. mRNA structural dynamics shape Argonaute-target interactions. Nat Struct Mol Biol 27, 790–801 (2020). https://doi.org/10.1038/s41594-020-0461-1

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