Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques
Uloženo v:
| Název: | Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques |
|---|---|
| Autoři: | Ombeline Pessey, Manikandan Karuppasamy, Isabel Chillón, Tina Uroda, Marco Marcia, Paolo Annibale, Jean-Marie Teulon, Jean-Luc Pellequer |
| Přispěvatelé: | Thomas, Frank, European Molecular Biology Laboratory Grenoble (EMBL), Max Delbrück Center for Molecular Medicine Berlin (MDC), Helmholtz-Gemeinschaft = Helmholtz Association, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), ISBG: UMS 3518 CNRS-CEA-UJF-EMBL, BM29-BioSAXS beamline, AFM platform at the IBS, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-15-CE11-0003,lincRNPs,Principes moleculaires de reconnaissance entre la proteine Polycomb SCML2A et longs ARN non-codants(2015), European Project: PJA-20191209284, European Project: ECTZ18552, European Project: 18CN047-00 |
| Zdroj: | Nature Protocols. 15:2107-2139 |
| Informace o vydavateli: | Springer Science and Business Media LLC, 2020. |
| Rok vydání: | 2020 |
| Témata: | 0301 basic medicine, 0303 health sciences, MESH: Microscopy, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Atomic Force, Microscopy, Atomic Force, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, MESH: Hydrodynamics, MESH: Image Processing, 03 medical and health sciences, Computer-Assisted, MESH: RNA, Hydrodynamics, Image Processing, Computer-Assisted, Long Noncoding, RNA, Long Noncoding |
| Popis: | 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. |
| Druh dokumentu: | Article |
| Jazyk: | English |
| ISSN: | 1750-2799 1754-2189 |
| DOI: | 10.1038/s41596-020-0323-7 |
| Přístupová URL adresa: | https://pubmed.ncbi.nlm.nih.gov/32451442 https://www.ncbi.nlm.nih.gov/pubmed/32451442 https://europepmc.org/article/MED/32451442 https://www.nature.com/articles/s41596-020-0323-7 https://www.nature.com/articles/s41596-020-0323-7.pdf https://www.scilit.net/article/b5b64d2fa402f26d52d0e71df42a520f https://pubmed.ncbi.nlm.nih.gov/32451442/ https://hal.univ-grenoble-alpes.fr/hal-03518165v1 https://doi.org/10.1038/s41596-020-0323-7 |
| Rights: | Springer TDM CC BY |
| Přístupové číslo: | edsair.doi.dedup.....f60008f0786b5cf33073a73554f99d46 |
| Databáze: | OpenAIRE |
Full Text Finder 
Nájsť tento článok vo Web of Science | Abstrakt: | 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. |
|---|---|
| ISSN: | 17502799 17542189 |
| DOI: | 10.1038/s41596-020-0323-7 |