Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling

Mitochondria contain the genetic information and expression machinery to produce proteins essential for cellular respiration. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins, and mitoribosome assembly factors are known to form punctate subcompartments referred to as mitochondrial RNA granules (MRGs) 1–3. Despite their proposed role in regulating gene expression, little is known about the structural and dynamic properties of MRGs. We investigated the organization of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy techniques, obtaining ultrastructural details of their internal architecture. We find that MRGs are organized into nanoscale RNA cores surrounded by a protein shell. Using live-cell super-resolution structured illumination microscopy and photobleaching perturbations, we reveal that MRGs undergo fusion and rapidly exchange components, consistent with liquid-liquid phase separation (LLPS). Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with membrane remodeling. Inhibition of mitochondrial fission leads to an aberrant distribution of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our results reveal a role for LLPS in concentrating RNA and its processing proteins into MRGs, which are positioned along mitochondria by membrane dynamics.

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Parallel monitoring of mRNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

10.1101/2020.07.14.203562 ◽

2020 ◽

Author(s):

Susanne Kramer ◽

Elisabeth Meyer-Natus ◽

Hanna Thoma ◽

Achim Schnaufer ◽

Markus Engstler

Keyword(s):

Single Molecule ◽

Spatial Information ◽

Electron Tomography ◽

In Situ Hybridisation ◽

Small Subset ◽

Mitochondrial Rna ◽

Fluorescence In Situ Hybridisation ◽

Tertiary Structures ◽

Rna Granules ◽

Lr White

ABSTRACTSingle mRNA molecules are frequently detected by single molecule fluorescence in situ hybridisation (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs.To overcome these limitations, we have hybridised slices of high pressure frozen, LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localisation that can be complemented with immunofluorescence and electron microscopy, as well as electron tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA tertiary structures, and we show that the method can be employed to probe for mRNA compactness. We apply LR White smFISH to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing, using trypanosomes and their versatile RNA biology as a model system.

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Mitochondrial RNA granules are fluid condensates positioned by membrane dynamics

Nature Cell Biology ◽

10.1038/s41556-020-00584-8 ◽

2020 ◽

Vol 22 (10) ◽

pp. 1180-1186 ◽

Cited By ~ 2

Author(s):

Timo Rey ◽

Sofia Zaganelli ◽

Emilie Cuillery ◽

Evangelia Vartholomaiou ◽

Marie Croisier ◽

...

Keyword(s):

Membrane Dynamics ◽

Mitochondrial Rna ◽

Rna Granules

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Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression

The Journal of Cell Biology ◽

10.1083/jcb.201507125 ◽

2016 ◽

Vol 212 (6) ◽

pp. 611-614 ◽

Cited By ~ 49

Author(s):

Alexis A. Jourdain ◽

Erik Boehm ◽

Kinsey Maundrell ◽

Jean-Claude Martinou

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Mitochondrial Gene ◽

Regulation Of Gene Expression ◽

Rna Degradation ◽

Mitochondrial Rna ◽

Mitochondrial Gene Expression ◽

Rna Granules ◽

Mitochondria Dna ◽

Replication Gene

In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized “mitochondrial RNA granules,” mitochondrial subdomains with an emerging role in the regulation of gene expression.

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High resolution imaging of nascent mitochondrial protein synthesis in cultured human cells

10.1101/2020.05.05.076109 ◽

2020 ◽

Author(s):

Matthew Zorkau ◽

Christin A Albus ◽

Rolando Berlinguer-Palmini ◽

Zofia MA Chrzanowska-Lightowlers ◽

Robert N. Lightowlers

Keyword(s):

Protein Synthesis ◽

Outer Membrane ◽

Mitochondrial Protein ◽

Human Cells ◽

Mitochondrial Rna ◽

Mitochondrial Protein Synthesis ◽

Inner Mitochondrial Membrane ◽

Rna Granules ◽

Cultured Human Cells ◽

Boundary Membrane

AbstractHuman mitochondria contain their own genome, mtDNA, that is expressed in the mitochondrial matrix. This genome encodes thirteen vital polypeptides that are components of the multi-subunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially-encoded components are also integral members of these complexes, where does nascent protein synthesis occur? Transcription, mRNA processing, maturation and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites ? We have adapted a click chemistry based method, coupled with STED nanoscopy to address these questions. We report that in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis occurs at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.

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Intracellular Targeting of Calmodulin mRNAs in Primary Hippocampal Cells

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540305100416 ◽

2003 ◽

Vol 51 (4) ◽

pp. 541-544 ◽

Cited By ~ 8

Author(s):

Elod Kortvely ◽

Szilvia Varszegi ◽

Arpad Palfi ◽

Karoly Gulya

Keyword(s):

In Situ Hybridization ◽

Glial Cells ◽

Cultured Cells ◽

Intracellular Distribution ◽

Low Level ◽

Rna Granules ◽

Intracellular Targeting ◽

Dendritic Mrna ◽

Mrna Targeting

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.

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