دورية أكاديمية
Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes
| العنوان: | Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes |
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| المؤلفون: | Smirnova, Anna, Jeandard, Damien, Smirnov, Alexandre |
| المساهمون: | Génétique moléculaire, génomique, microbiologie (GMGM), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut d’Etudes Avancées de l’Université de Strasbourg - Institute for Advanced Study (USIAS), Université de Strasbourg (UNISTRA), IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT_US project (ANR 20-SFRI-0012), EUR IMCBio (ANR17-EURE-0023), IdEx – Attractivité (ANR-10-IDEX-0002-02) |
| المصدر: | ISSN: 2331-8325 ; Bio-protocol ; https://hal.science/hal-04234522Test ; Bio-protocol , 2023, 13 (18), pp.e4820. ⟨10.21769/BioProtoc.4820⟩ ; https://en.bio-protocol.org/en/bpdetail?id=4820&type=0Test. |
| بيانات النشر: | HAL CCSD Bio-protocol LCC |
| سنة النشر: | 2023 |
| المجموعة: | Archive ouverte HAL (Hyper Article en Ligne, CCSD - Centre pour la Communication Scientifique Directe) |
| مصطلحات موضوعية: | CoLoC-seq, Subcellular transcriptomics, RNA localisation, Cell fractionation, RNase, Enzymatic kinetics, Nonlinear regression, Northern blotting, Membrane-bounded organelle, Mitochondria, CoLoC-seq Subcellular transcriptomics RNA localisation Cell fractionation RNase Enzymatic kinetics Nonlinear regression Northern blotting Membrane-bounded organelle Mitochondria, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology |
| الوصف: | International audience ; Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host–pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| العلاقة: | info:eu-repo/semantics/altIdentifier/pmid/37753469; hal-04234522; https://hal.science/hal-04234522Test; https://hal.science/hal-04234522/documentTest; https://hal.science/hal-04234522/file/Smirnova%20%26%20al.%20%282023%29%20Bio-protocol.pdfTest; PUBMED: 37753469 |
| DOI: | 10.21769/BioProtoc.4820 |
| الإتاحة: | https://doi.org/10.21769/BioProtoc.4820Test https://hal.science/hal-04234522Test https://hal.science/hal-04234522/documentTest https://hal.science/hal-04234522/file/Smirnova%20%26%20al.%20%282023%29%20Bio-protocol.pdfTest |
| حقوق: | http://creativecommons.org/licenses/by-ncTest/ ; info:eu-repo/semantics/OpenAccess |
| رقم الانضمام: | edsbas.F9F19EBE |
| قاعدة البيانات: | BASE |
| DOI: | 10.21769/BioProtoc.4820 |
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