Abstract
Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3–4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts.
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Data availability
All data generated or analyzed during this study are included in the paper and its Supplementary Information and are available from the corresponding author on request. We have deposited in figshare an extensive set of AFM images, which we have used for calculation of the PSDs:
MEG3 v1 compact form: https://figshare.com/s/e73aff439aeaad75e456
MEG3 v1 intermediate form: https://figshare.com/s/a81600774ab9baed8932
MEG3 v1 denatured form: https://figshare.com/s/026d8eb31a8a4b6910e9
MEG3 H11LpA mutant, compact form: https://figshare.com/s/666c9998f22d661d72f3
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Acknowledgements
We thank A. McCarthy and the beamline scientists at the BM29-BioSAXS beamline for their support during SAXS data collection; A. Leroy and C. Ebel (IBS Grenoble) for support with AUC; S. Acajjaoui and M. Soler Lopez (ESRF Grenoble) for support with DLS; and C. Mas (IBSG Grenoble) and M. Jamin (UGA Grenoble) for support with SEC-MALLS. We also thank all members of the Marcia lab for helpful discussions. Work in the Marcia lab was partly funded by the Agence Nationale de la Recherche (ANR-15-CE11-0003-01), the Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS, ECTZ18552), ITMO Cancer (18CN047-00), and the Fondation ARC pour la recherche sur le cancer (PJA-20191209284). This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work acknowledges the AFM platform at the IBS.
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M.M. conceived the project and obtained funding; M.M. and I.C. supervised the research and wrote the initial draft of the manuscript; T.U., M.M., J.-M.T., and J.-L.P. developed the procedures for AFM image acquisition; T.U., I.C., and M.M. developed the procedures for lncRNA purification, AUC, and MALLS; T.U. and M.M. developed the procedures for DLS and SAXS experiments; M.K. carried out EM experiments; O.P. provided technical assistance and contributed to sample preparation; P.A., J.-L.P., and M.M. developed the procedures for AFM image analysis. All authors reviewed and edited the manuscript and approved the final draft.
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Peer review information Nature Protocols thanks Yuri L. Lyubchenko, Neil H. Thomson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Uroda, T. et al. Mol. Cell 75, 982–995 (2019): https://doi.org/10.1016/j.molcel.2019.07.025
Extended Data
Extended Data Fig. 1 Negative-staining electron microscopy and atomic force microscopy.
(a) Raw micrograph displaying individual MEG3 particles in 17.5 mM Mg2+ stained with uranyl formate. Aggregates of different size are visible in the image. The scale bar of 100 nm is reported at the top right corner of the panel. (b) Reference-free 2d class averages of single particles selected from the same micrographs as reported in panel A. The scale bar of 30 nm is reported at the top right of the panel. (c) Overview of the mica surface spotted with MEG3 particles in the folded state (scanning resolution = 9.8 nm/pixel). The scale bar of 2 µm is reported at the top right corner of the panel. This panel was adapted from Uroda et al10. (d) Topographical AFM image without particle averaging at a scanning resolution of 0.98 nm/pixel. The scale bar of 400 nm is reported at the top right corner of the panel. This panel is reproduced from Fig. 4, compact form of MEG3.
Extended Data Fig. 2 Cryo-electron microscopy.
Representative micrographs showing MEG3 aggregates as seen on the carbon support of cryo-EM holey grids (in 25 mM Mg2+). The scale bars of 100 nm are reported at the top right corner of each panel.
Extended Data Fig. 3 Denaturing versus non-denaturing purifications of lncRNAs.
(a) SEC-MALLS profiles of MEG3 v1 purified under the non-denaturing conditions described in this protocol. Adapted from Uroda et al10 under a Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). (b) SEC-MALLS of MEG3 v1 purified under denaturing conditions and then refolded using a slow temperature gradient. (c) SEC-MALLS of MEG3 v1 purified under denaturing conditions and then refolded using a fast temperature gradient. (d) DLS profiles of MEG3 v1 purified under non-denaturing conditions or under denaturing conditions and slow refolding. Denaturation and refolding inevitably cause sample heterogeneity.
Extended Data Fig. 4 Hydrodynamic properties of lncRNAs in magnesium-containing buffers.
(a) Representative raw data distribution and residual determined in SEDFIT for MEG3 v1 in 0.1 mM MgCl2. This panel is also represented as an icon in Fig. 1. (b) Sedimentation velocity AUC profiles of MEG3 v1 at the indicated magnesium concentrations. The black arrow indicates signal from a small population of multimeric species or aggregates that form at high magnesium concentrations (> 10 mM). (c) Hill curve representing the Rh distribution at different magnesium concentrations for MEG3 v1. This panel is modified from Uroda et al10. (d) SEC-MALLS profiles of MEG3 v1 eluted in the absence of magnesium and in the presence of 10 mM magnesium. In the latter condition, the aggregates visible by AUC (panel A) compromise the light scattering profile. The panel representing the sample in the absence of magnesium is the same as in Extended Data Fig. 3a. (e) Native agarose gel electrophoresis of MEG3 v1 at the indicated magnesium concentrations (in mM, on the top of each lane). d,e, adapted from Uroda et al10 under a Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode).
Extended Data Fig. 5 AFM troubleshooting.
Under suboptimal conditions salt crystals or particle aggregates tend to form on the AFM support. (a) Sample dried too long. The scale bar of 400 nm is reported at the top right corner of the panel. (b) Sample washed insufficiently (50 µL of DEPC-treated water). The scale bar of 400 nm is reported at the top center of the panel. (c) Sample denatured using urea. The scale bar of 400 nm is reported at the top right corner of the panel. (d) Sample deposited on non-derivatized mica (left) vs on mica derivatized with APTES (right). The scale bar of 400 nm is reported at the bottom left corner of each panel.
Extended Data Fig. 6 Quality control of lncRNA purity and homogeneity.
(a) Agarose gel electrophoresis of different transcription reactions of MEG3 v1. M indicate the Quick load 2-log DNA molecular size marker; 1-4 are samples from initial transcription screenings, immediately after transcription (Step 5; 1-4 indicate the transcription buffer used, see Tables 2 and 3); D is a degraded sample; P is a sample transcribed in buffer 4 after SEC (Step 18). The arrows on the right indicate the position of MEG3 and of the linearized transcription vector, which is occasionally visible in samples that have not yet been subjected to DNase treatment, but disappears after purification. (b) Representative Bioanalyzer run of MEG3 v1 transcribed from buffer 4. M is the molecular size marker provided in the Agilent RNA 6000 Nano Kit.
Extended Data Fig. 7 SAXS analysis of lncRNAs.
(a) Scattering curve, (b) Guinier plot, and (c) P(r) plot for MEG3 v1. a–c adapted from Uroda et al10 under a Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). (d) DAMMIF ab initio shape for MEG3 v1. Damaver.pdb output represented as a blue mesh, damfilt.pdb output represented as an orange solid surface (see Step 64 for a description of these files).
Supplementary information
Source data
Source Data Fig. 2
Unprocessed AFM image.
Source Data Fig. 3
Unprocessed AFM image.
Source Data Fig. 4
Unprocessed AFM images.
Source Data Extended Data Fig. 1
Unprocessed AFM and EM images
Source Data Extended Data Fig. 2
Unprocessed EM images.
Source Data Extended Data Fig. 4
Unprocessed agarose gel scans.
Source Data Extended Data Fig. 5
Unprocessed AFM images.
Source Data Extended Data Fig. 6
Unprocessed agarose gel scans.
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Uroda, T., Chillón, I., Annibale, P. et al. Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques. Nat Protoc 15, 2107–2139 (2020). https://doi.org/10.1038/s41596-020-0323-7
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DOI: https://doi.org/10.1038/s41596-020-0323-7
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