Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy

  • The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.
Author:Sina MangerORCiD, Utz Heinrich ErmelORCiD, Achilleas S. FrangakisORCiDGND
Parent Title (English):Communications biology
Publisher:Springer Nature
Place of publication:London
Document Type:Article
Date of Publication (online):2021/02/19
Date of first Publication:2021/02/19
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/01/17
Tag:Cryoelectron microscopy; Gene expression analysis; Genetic engineering
Issue:art. 234
Article Number:234
Page Number:8
First Page:1
Last Page:8
This work was funded by the Deutsche Forschungsgemeinschaft grants FR1653/12 and FR1653/14 as well as SFB902 (project B5). Open Access funding enabled and organized by Projekt DEAL.
Exzellenzcluster / Exzellenzcluster Makromolekulare Komplexe
Fachübergreifende Einrichtungen / Buchmann Institut für Molekulare Lebenswissenschaften (BMLS)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoCreative Commons - Namensnennung 4.0