Abstract
The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Iborra FJ, Kimura H, Cook PR (2004) The functional organization of mitochondrial genomes in human cells. BMC Biol 2(1):9
Jourdain A, Koppen M, Wydro M, Rodley C, Lightowlers R, Chrzanowska-Lightowlers Z, Martinou J (2013) GRSF1 regulates RNA processing in mitochondrial RNA granules. Cell Metab 17(3):399–410
Antonicka H, Sasarman F, Nishimura T, Paupe V, Shoubridge E (2013) The mitochondrial RNA-binding protein GRSF1 localizes to RNA granules and is required for posttranscriptional mitochondrial gene expression. Cell Metab 17(3):386–398
Jourdain A, Koppen M, Rodley C, Maundrell K, Gueguen N, Reynier P, Martinou J (2015) A mitochondria-specific isoform of FASTK is present in mitochondrial RNA granules and regulates gene expression and function. Cell Rep 10(7):1110–1121
Antonicka H, Shoubridge E (2015) Mitochondrial RNA granules are centers for posttranscriptional RNA processing and ribosome biogenesis. Cell Rep 10(6):920–932
Tu Y, Barrientos A (2015) The human mitochondrial DEAD-box protein DDX28 resides in RNA granules and functions in mitoribosome assembly. Cell Rep 10(6):854–864
Jourdain AA, Boehm E, Maundrell K, Martinou J (2016) Mitochondrial RNA granules: compartmentalizing mitochondrial gene expression. J Cell Biol 212(6):611–614
Jao CY, Salic A (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. PNAS 105(41):15779–15784
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Xavier, V.J., Martinou, JC. (2021). Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling. In: Minczuk, M., Rorbach, J. (eds) Mitochondrial Gene Expression. Methods in Molecular Biology, vol 2192. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0834-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0834-0_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0833-3
Online ISBN: 978-1-0716-0834-0
eBook Packages: Springer Protocols