DOI: 10.5061/dryad.rxwdbrvkt
title: Data from: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

description: Protein- and RNA-rich bodies contribute to the spatial organization of
 gene expression in the cell and are also sites of quality control critical
 to cell fitness. In most eukaryotes, mitochondria harbor their own genome,
 and all steps of mitochondrial gene expression co-occur within a single
 compartment – the matrix. Here, we report that processed mitochondrial
 RNAs are consolidated into micron-scale translation hubs distal to
 mitochondrial DNA transcription and RNA processing sites in human cells.
 We find that, during stress, mitochondrial messenger and ribosomal RNA is
 sequestered in mesoscale bodies containing mitoribosome components,
 concurrent with suppression of active translation. Stress bodies are
 triggered by proteotoxic stress downstream of double-stranded RNA
 accumulation in cells lacking unwinding activity of the highly conserved
 helicase SUPV3L1/SUV3. We propose that the spatial organization of nascent
 polypeptide synthesis into discrete domains serves to throttle the flow of
 genetic information to support recovery of mitochondrial quality control.
description: This dataset contains raw data and analyses used to produce
 figures in the referenced manuscript by Begeman, et al. A detailed
 description of the methods can be found in the manuscript. Additionally, a
 stepwise protocol for labeling cells with key reagent
 L-Homopropargylglycine is available in the protocols.io repository via the
 following doi:
 https://dx.doi.org/10.17504/protocols.io.3byl4wedrvo5/v1.
 Raw microscopy data are provided as 16-bit .CZI files that are
 native to Carl Zeiss Microscopy ZEN software version 3.7 or higher, or as
 TIFF files. CZI files can also be opened with ImageJ/Fiji with the
 Bioformats plugin. Data analysis on which manuscript
 figures are based is provided as: csv files, text files, Prism analysis
 files, or Microsoft Excel spreadsheets.
description: # Data from: Spatial analysis of mitochondrial gene expression reveals
 dynamic translation hubs and remodeling in stress ## General Information
 ### Dataset Overview For a comprehensive overview and the methodology used
 to generate and process all the data in this repository please see the
 corresponding open access publication (see below). ### Corresponding
 Author Information ``` Name: Samantha C Lewis ORCID:
 https://orcid.org/0000-0001-6306-443X Affiliations: Department of
 Molecular and Cell Biology, University of California, Berkeley, CA USA
 Innovative Genomics Institute, Berkeley, CA, USA Helen Wills Neuroscience
 Institute, Berkeley, CA USA Department of Nutritional Sciences and
 Toxicology, University of California, Berkeley, CA USA email:
 samlewis@berkeley.edu ``` ### Related Publication ``` Begeman, A., Smolka,
 J. A., Shami, A., Pradip Waingankar, T., Lewis, S. C., Spatial analysis of
 mitochondrial gene expression reveals dynamic translation hubs and
 remodeling in stress.Sci. Adv.11,eads6830(2025).DOI:10.1126/sciadv.ads6830
 ``` ### Funding Information ``` This work was supported by a NSF Graduate
 Research Fellowship to AB, and N.I.H. grants R00GM129456 and R35GM147218
 to SL. The content is solely the responsibility of the authors and does
 not necessarily represent the official views of the U.S. National
 Institutes of Health or the U.S. National Science Foundation. ``` ## Notes
 for Files ### Image Names When applicable, image file names follow the
 pattern: `(condition)_{(protein being targeted/ fluorescent object)-(laser
 line)}x2-4.czi` ### Microscopy and Image Acquisition All images were
 acquired using a Zeiss LSM 980 with Airyscan 2 laser scanning confocal
 microscope, equipped with 405-, 588-, 561-, and 639-nm laser lines and
 Fast Airyscan detector array. Images were acquired using an inverted
 63×/1.4 numerical aperture oil objective. All live imaging was done in a
 humidified chamber at 37°C and in the presence of 5% CO2. Airyscan
 processing and maximum intensity image projection were performed using
 Zeiss ZEN Blue software version 3.7 (Carl Zeiss). Image brightness and/or
 contrast were linearly adjusted in ZEN Blue or FIJI. ### File Types *
 .czi: Zeiss raw microscopy image file type. * .roi: FIJI region of
 interest file. * .txt: plain text file showing image metadata. * .csv:
 data file for tabular data. * .xlsx: excel data file for tabular data. *
 .pipeline: Zeiss Arivis image analysis pipeline file type. * .training:
 Zeiss Arivis machine learning image segmentation file type. * .py: Python
 files for custom analysis code. * .ijm.py: FIJI macro file in the python
 language. ### Software Used and Python Packages Required * Zeiss Zen Blue
 Version 3.7 * Arivis Vision4D Version 4.1 * ImageJ (FIJI) Version 1.54f *
 Python 3.8 * scipy v.1.4.1 * pandas v.1.3.3 * matplotlib v.3.1.2 * numpy
 v.1.21.2 * seaborn v.0.10.1 * scikit-learn v.1.0.1 * Microsoft Excel ##
 Description of Directories and File Structure ###
 *Begemanetal\_Sci\_Adv\_2025\_Raw\_Data.zip* The compressed master
 directory contains 3 sub-directories corresponding to the code used to
 generate figures and analysis for the paper, images and data used in the
 main text figures, and images and data used in the supplemental figures.
 ``` Begemanetal_Sci_Adv_2025_Raw_Data |- Code used in paper/ |- Main
 Figure Raw Data/ |- Supplemental Figure Raw Data/ ``` ### Sub-directories
 #### *Code used in paper* Custom code generated for the analysis and
 quantification used in the paper. Sub-directories separated by software or
 environment. ``` |- Begeman_et_al_Arivis_Code/ |- Begeman_et_al_FIJI_Code/
 |- Bgeman_et_al_Python_Code/ ``` ##### Begeman\_et\_al\_Arivis\_Code
 Arivis pipeline files and trainings for machine learning based
 segmentation. Detailed workflow can be found in the corresponding
 publication (see above), and outputs and files used for each pipeline
 shown in the figures and quantification for main and supplemental data
 section below. * ML Training: Machine learning training files used in the
 segmentation for Arivis pipeline files. * EU GRSF1 Pipeline.pipeline:
 Arivis pipeline file for segmenting EU and GRSF1 in mitochondria. * GRSF1
 and DNA Pipeline v2.pipeline: Arivis pipeline file for segmenting GRSF1
 and dsDNA in mitochondria. * HPG CAP and 15 min Pulse Control
 Pipeline.pipeline: Arivis pipeline file for segmenting HPG in
 mitochondria. * HPG IMT1B Control Pipeline.pipeline: Arivis pipeline file
 for segmenting HPG in mitochondria treated with IMT1b. * HPG with GRSF1
 Pipeline.pipeline: Arivis pipeline file for segmenting HPG and GRSF1 in
 mitochondria. * HPG with MRPL23 Pipeline.pipeline: Arivis pipeline file
 for segmenting HPG and MRPL23 in mitochondria. * HPG with TFAM
 Pipeline.pipeline: Arivis pipeline file for segmenting HPG and TFAM in
 mitochondria. * image mask save 4 channel.pipeline: Arivis pipeline file
 for creating image mask of mitochondria for image with 4 channels. * image
 mask save SUV3-HA.pipeline: Arivis pipeline file for creating image mask
 for image set of SUV3-HA expressing cells. * image mask save.pipeline:
 Arivis pipeline file for creating image mask of mitochondria for images
 with 3 channels. * mtRNA Glut Fix Pipeline.pipeline: Arivis pipeline file
 for segmenting mt-RNA and dsDNA in mitochondria. * ND4 (Blob) and MRPL23
 Pipeline.pipeline: Arivis pipeline file for segmenting ND4 and MRPL23 in
 mitochondria. * ND4 (Blob) GRSF1 dsDNA Pipeline.pipeline: Arivis pipeline
 file for segmenting ND4, dsDNA, and GRSF1 in mitochondria. * ND4 HPG
 Pipeline (Blob).pipeline: Arivis pipeline file for segmenting ND4 and HPG
 in mitochondria. * New HPG Segmentation Pipeline 1000 HPG
 threshold.pipeline: Arivis pipeline file for segmenting HPG in
 mitochondria using an intensity threshold of 1000. * New HPG Segmentation
 Pipeline 2.pipeline: Arivis pipeline file for segmenting HPG in
 mitochondria. * Reverse Image Mask.pipeline: Arivis pipeline file for
 creating a reverse mitochondrial image mask. * RNR2 and MRPL23
 Pipeline.pipeline: Arivis pipeline file for segmenting RNR2 and MRPL23 in
 mitochondria. * RNR2 GRSF1 dsDNA Pipeline.pipeline: Arivis pipeline file
 for segmenting dsDNA, GRSF1, and RNR2 in mitochondria. * RNR2 HPG DRP1K38A
 Pipeline v4.pipeline: Arivis pipeline file for segmenting HPG and RNR2 in
 mitochondria, and separating DRP1K38A expressing cells from those not. *
 RNR2 HPG Pipeline v2.pipeline: Arivis pipeline file for segmenting RNR2
 and HPG in mitochondria. * SUV3 Cat Dead Rescue Pipeline.pipeline: Arivis
 pipeline file for segmenting mt-RNA and HA signal in mitochondria in
 SUV3KO cells expressing a cat dead mutant of SUV3. * SUV3 KO HPG Mito Only
 Analysis.pipeline: Arivis pipeline file for segmenting HPG in mitochondria
 of SUV3 KO cells. * SUV3 WT Rescue Pipeline First Bio.pipeline: Arivis
 pipeline file for segmenting mt-RNA and HA signal in mitochondria in
 SUV3KO cells expressing WT SUV3. * SUV3-HA GRSF1 Pipeline.pipeline: Arivis
 pipeline file for segmenting HA signal and GRSF1 in mitochondria. * SUV3KO
 RNR2 Posttreatment Pipeline v3.pipeline: Arivis pipeline file for
 segmenting mt-RNA in mitochondria of SUV3KO cells treated with CAP after
 KO. * SUV3KO RNR2 Pretreatment Pipeline v3.pipeline: Arivis pipeline file
 for segmenting mt-RNA in mitochondria of SUV3KO cells treated with CAP
 before KO. * TFAM and DNA Pipeline.pipeline: Arivis pipeline file for
 segmenting TFAM and dsDNA in mitochondria. * tRNAs and MRPL23
 Pipeline.pipeline: Arivis pipeline file for segmenting tRNAs and MRPL23 in
 mitochondria. * tRNAs GRSF1 dsDNA Pipeline.pipeline: Arivis pipeline file
 for segmenting tRNAs, GRSF1, and dsDNA in mitochondria. * tRNAs HPG
 Pipeline v2.pipeline: Arivis pipeline file for segmenting tRNAs and HPG in
 mitochondria. ##### Begeman\_et\_al\_FIJI\_Code FIJI python macros for
 pulling Kymograph data and for the skeletonization and line scan
 extraction used in the automated line scan analysis. Detailed workflow can
 be found in the corresponding publication (see above). *
 PullKymographData.ijm.ijm.py: FIJI macro file for pulling kymograph data
 from open movie. * SkeletontoLineScan.ijm.ijm.py: FIJI macro file for
 skeletonizing mitochondria and pulling linescans from the skeletons.
 Expects masked mitochondria and returns csvs of linescans. #####
 Begeman\_et\_al\_Python\_Code Custom python code used to generate
 automated line scan analysis, object intersection data, and to compile
 other quantifications throughout the paper. Detailed workflow can be found
 in the corresponding publication (see above). * AvgLineScan: python files,
 csv inputs, and csv outputs for generating average line scan profiles from
 thousands of individual fluorescence line scans. *
 Visual_lineScan_test.py: python file for visualizing individual linescans
 and picking correct peak calling parameters. * pull_avg_linescan.py:
 python file for generating average linescan profile from thousands of
 individual linescans centered around specific peak. *
 pull_individual_peaks_and_cluster.py: python file for clustering
 individual linescan peaks. * pull_linescan_confidence.py: python file for
 calculating confidence values for average linescan profile. *
 pull_random_data_peaks.py: python file that generates average linescan
 profile centered around random positions along the linescan rather than a
 fluorescence peak. * Normalization_calc_v2.xlsx: Excel spreadsheet used to
 calculate the normalized average linescan profile. * Directories of input
 linescans and output csvs from the python files. * DRP1K38A Analysis:
 python files, csv inputs, and csv outputs corresponding to DRP1K38A
 quantification. * Hand_DRP1K38A_Analysis.py: python file used to aggregate
 data from `hand_csvs_v2/` csvs generated from Arivis segmentation into
 `hand_agg_analysis_v2.csv` * HPGControls: python files, csv inputs, and
 csv outputs corresponding to HPG controls quantification. * CAP_Control:
 Directory of input csvs from CAP HPG Arivis segmentation, python file
 aggregating data into output file. * IMT1B_Control: Directory of input
 csvs from IMT1B HPG Arivis segmentation, python file aggregating data into
 output file. * mtRNAControls: Python files, csv inputs, and csv outputs of
 quantification of mt-RNA abundance in Arivis segmented mitochondria in all
 specified conditions. * ObjectIntersections: * datasets: directories of
 objects of interest with python files used to aggregate object
 intersection data from Arivis segmentation. * run_object_simulation:
 custom python package containing `object_intersection_sim.py` a custom
 python script that calculates the percentage of the time two objects of
 given sizes, abundance, and restricted by a set volume will intersect by
 random chance through a monte Carlo simulation. * SUV3Analysis: Python
 files, csv inputs, and csv outputs of quantification of SUV3 KO cells used
 below. * ChloroTreatment: python files, csv inputs, and csv outputs of
 quantification of mitochondrial stress body sizes in SUV3KO cells pre and
 post treated with CAP. * HPG: python files, csv inputs, and csv outputs of
 quantification of HPG intensity in mitochondria of SUV3KO cells. * Rescue:
 python files, csv inputs, and csv outputs of quantification of
 mitochondrial stress body sizes/ prevalence in SUV3 rescue conditions
 discussed below. #### *Main Figure Raw Data* All images and data used to
 generate the main figures. Sub-directories are separated by figure number
 and further separated by their corresponding panel of that figure. The
 image shown in the figure as well as its metadata are included for each
 panel and the images that went into the given analysis are located in the
 corresponding folder named Images Analyzed. Raw csv files for the shown
 quantification are given as well with variable names corresponding to the
 respective figure and condition. ``` |---Figure 01 |---Figure 01B
 |---Figure 01C |---Figure 01D |---Figure 01E |---Figure 01F-G-H
 |---01F-G-H Images Analyzed |---Figure 02 |---Figure 02B |---Figure 02C
 |---02C Images Analyzed |---Figure 02D |---02D Images Analyzed |---Figure
 02E |---Figure 02F |---02F Images Analyzed |---Figure 02G-H |---02G-H
 Images Analyzed |---Figure 03 |---Figure 03A |---Figure 03B-C-D
 |---03B-C-D Images Analyzed |---Figure 03E |---Figure 03F-G-H |---03F-G-H
 Images Analyzed |---Figure 03I |---Figure 03J-K-L-M |---03J-K-L-M Images
 Analyzed |---Figure 04 |---Figure 04B |---Figure 04C |---Figure 05
 |---Figure 05A |---Figure 05B |---05E Images Analyzed |---Figure 06
 |---Figure 06A |---06A Images Analyzed |---Figure 06B |---06B Images
 Analyzed |---Figure 06C |---Figure 06D |---Figure 06E ``` ##### Figure 01
 Images and quantification showing that processed mtRNA is excluded from
 nucleoids and MRGs. * Figure 01B: * Representative image and image
 metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20
 (c2), dsDNA (c3), and RNA-FISH targeting mitoribosomal component RNR2
 (c1). * CSV file of fluorescent intensities for each imaging channel
 listed above across a representative linescan distance. * Figure 01C: *
 Representative image and image metadata of fixed IMR90 cells immunolabeled
 with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH targeting
 mitochondrial tRNAs (c1). * CSV file of fluorescent intensities for each
 imaging channel listed above across a representative linescan distance. *
 Figure 01D: * Representative image and image metadata of fixed IMR90 cells
 immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH
 targeting ND4 mRNA (c1). * CSV file of fluorescent intensities for each
 imaging channel listed above across a representative linescan distance. *
 Figure 01E: * Representative image and image metadata of fixed IMR90 cells
 immunolabeled with antibodies against GRSF1 (c2), dsDNA (c3), and RNA-FISH
 targeting ND4 mRNA (c1). * Figure 01F-G-H: * 01F-G-H Images Analyzed: All
 images used for the analysis of mt-RNA interactions with GRSF1 and dsDNA *
 ND4_GRSF1_dsDNA_Linescan_Analysis_Data.csv: Data for iterative linescan
 analysis of ND4 mRNA (c1), GRSF1 (c2), and dsDNA (c3). Data generated by
 first segmenting mitochondrial objects using `ND4 (Blob) GRSF1 dsDNA
 Pipline.pipeline` located in `Code used in
 Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro
 `SkeletontoLineScan.imj.imj.py` located in `Code used in
 paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual
 linescans of fluorescence intensity that were then used to generate
 average linescan using custom python code located at `Code used in
 Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column
 labels. * Distance: distance in microns * Headers follow pattern:
 C(channel number used for peak calling) Channel (channel of fluorescent
 intensity) * Mean: mean fluorescence intensity * SD: standard deviation in
 fluorescence intensity * N: number of individual linescans that go into
 iterative analysis * RNR2_GRSF1_dsDNA_Linescan_Analysis_Data.csv: Data for
 iterative linescan analysis of mitoribosomal RNA component RNR2 (c1),
 GRSF1 (c2), and dsDNA (c3). Data generated by first segmenting
 mitochondrial objects using `RNR2 GRSF1 dsDNA Pipline.pipeline` located in
 `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in
 Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in
 paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual
 linescans of fluorescence intensity that were then used to generate
 average linescan using custom python code located at `Code used in
 Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column
 labels. * Distance: distance in microns * Headers follow pattern:
 C(channel number used for peak calling) Channel (channel of fluorescent
 intensity) * Mean: mean fluorescence intensity * SD: standard deviation in
 fluorescence intensity * N: number of individual linescans that go into
 iterative analysis * tRNAs_GRSF1_dsDNA_Linescan_Analysis_Data.csv: Data
 for iterative linescan analysis of mitochondrial tRNAs (c1), GRSF1 (c2),
 and dsDNA (c3). Data generated by first segmenting mitochondrial objects
 using `tRNAs GRSF1 dsDNA Pipline.pipeline` located in `Code used in
 Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro
 `SkeletontoLineScan.imj.imj.py` located in `Code used in
 paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual
 linescans of fluorescence intensity that were then used to generate
 average linescan using custom python code located at `Code used in
 Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column
 labels. * Distance: distance in microns * Headers follow pattern:
 C(channel number used for peak calling) Channel (channel of fluorescent
 intensity) * Mean: mean fluorescence intensity * SD: standard deviation in
 fluorescence intensity * N: number of individual linescans that go into
 iterative analysis * Object_Intersection_Data.csv: Data for quantifying
 the interactions between ND4 mRNA, GRSF1, and dsDNA objects generated from
 `ND4 (Blob) GRSF1 dsDNA Pipeline.pipeline` located at `Code used in
 Paper/Begeman_et_al_Arivis_Code/` and compiled using custom python code
 located at `Code used in
 Paper/Begeman_et_al_Python_Code/ObjectIntersection/datasets/ND4_Blob_GRSF1/` with the following column labels. * sum of mito area: Total segmented mitochondrial area in images used for analysis. * number of (object): total number of given segmented object in images used for analysis. * sum of (object) area: total area of given segmented object in images used for analysis. * avg (object) area: avg area of the given segmented object in images used for analysis. * number of (object 1) in (object 2): total number of the given segmented object 1 overlapping with the given segmented object 2 in images used for analysis. * percent of (object 1) in (object 2): percent of the given segmented object 1 overlapping with the given segmented object 2 in images used for analysis. * avg percent of simulated (object 1) in (object 2): percentage of given object 1s that would overlap with object 2 by random chance given their respective abundance, sizes, and mitochondrial area. Generated using custom `object_intersection_sim.py` python file in `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/run_object_simulation/`. ##### Figure 02 Images and quantification showing that processed mtRNA marks local mitochondrial translation hubs. * Figure 02B: * Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), MRPL23 (c2), and RNA-FISH targeting ND4 mRNA (c1). * Figure 02C: * 02C Images Analyzed: All images used in the quantification of ND4 mRNA and MRPL23 object abundance and overlap. * Object_Intersection_Data.csv: Data for quantifying the interactions between ND4 mRNA and MRPL23 objects generated from `ND4 (Blob) and MRPL23 Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` and compiled using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/datasets/ND4_Blob_MRPL23/` with the following column labels. * number of ND4 per mito area: number of segmented ND4 objects normalized to segmented mitochondrial object area. * number of MRPL23 per mito area: number of segmented MRPL23 objects normalized to segmented mitochondrial object area. * percent of ND4 with MRPL23: percent of segmented ND4 objects overlapping with segmented MRPL23 objects. * percent of ND4 with MRPL23 Above Sim: percent of segmented ND4 objects overlapping with segmented MRPL23 objects above what is expected by random chance given their respective abundance, sizes, and mitochondrial area. Generated using custom `object_intersection_sim.py` python file in `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/run_object_simulation/`. * percent of MRPL23 with ND4: percent of segmented MRPl23 objects overlapping with segmented ND4 objects. * percent of MRPL23 with ND4 above Sim: percent of segmented MRPL23 objects overlapping with segmented ND4 objects above what is expected by random chance given their respective abundance, sizes, and mitochondrial area. Generated using custom `object_intersection_sim.py` python file in `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/run_object_simulation/`. * Figure 02D: * Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), MRPL23 (c2), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 02D Images Analyzed: All images used in the generation of the average linescan for MRPL23 and HPG. * Linescan_Analysis_Data.csv: Data for iterative linescan analysis of mitochondrial HPG (c1), and MRPL23 (c2). Data generated by first segmenting mitochondrial objects using `HPG with MRPL23 Pipeline.pipeline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * Figure 02E: * 15minPulse_HPG-647_TOM20-488_dsDNA-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 48hrDMSO_RNR2-647_HPG-488_TOM20-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitoribosomal component RNR2 (c1), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c2) first treated for 48hr with 0.1% DMSO. * 48hrIMT1B_ND4-647_HPG-488_TOM20-405-05-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against ND4 mRNA (c1), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c2) first treated for 48hr with 10uM IMT1B to inhibit mitochondrial transcription. * Chloro_HPG-647_TOM20-488_dsDNA-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-647 to show mitochondrial translation (c1) treated with 50 ug/uL chloramphenicol 20 minutes prior to HPG introduction to inhibit mitochondrial translation. * Figure 02F: * 02F Images Analyzed: All images used in the quantification of HPG intensity in control mitochondria or those treated with 10uM IMT1b 48hr prior to HPG labeling, or 50ug/uL chloramphenicol 20 minutes prior to HPG labeling. * HPG_Quantification.csv: Data for average fluorescence intensity in segmented mitochondrial objects generated from `HPG CAP and 15 min Pulse Control Pipeline.pipeline` and `HPG IMT1B Control Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` and compiled using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/HPGControls/` with the following column labels. * 15 min Pulse: Control cells labeled with HPG normally * CAP: cells treated with 50 ug/uL chloramphenicol 20 minutes prior to HPG labeling. * DMSO: Control cells for IMT1b condition treated with 0.1% DMSO for 48hr prior to HPG labeling. * IMT1B: cells treated with 10 uM IMT1b for 48hr prior to HPG labeling. * Figure 02G-H: * (RNA Species)-647_L-HPG-488_TOM20-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against either mitoribosomal component RNR2, mitochondrial tRNAs, or ND4 mRNA (c1), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c2). * 02G-H Images Analyzed: All images used in the quantification of mitochondrial RNA intensity overlapping with HPG intensity. * (RNA Species)_HPG_TOM20_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of either mitoribosomal RNA RNR2, mitochondrial tRNAs, or ND4 mRNA (c1), HPG (c2), and TOM20 (c3) Data generated by first segmenting mitochondrial objects using respective `RNR2 HPG Pipeline v2.pipline`, `tRNAs HPG Pipeline v2.pipline` or `ND4 HPG Pipeline (Blob).pipline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * (RNA Species)_HPG_TOM20_Object_Intersection_Data.csv: Data for quantifying the interactions between either mitoribosomal RNA RNR2, mitochondrial tRNAs, or ND4 mRNA objects and HPG objects generated from corresponding `RNR2 HPG Pipeline v2.pipline`, `tRNAs HPG Pipeline v2.pipline` or `ND4 HPG Pipeline (Blob).pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` and compiled using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/datasets/` and either `RNR2_HPG_v2/`, `tRNAs_HPG_v2/`, or `ND4_Blob_HPG/` with the following column labels. * sum of mito area: Total segmented mitochondrial area in images used for analysis. * number of (object): total number of given segmented object in images used for analysis. * sum of (object) area: total area of given segmented object in images used for analysis. * avg (object) area: avg area of the given segmented object in images used for analysis. * number of (object 1) in (object 2): total number of the given segmented object 1 overlapping with the given segmented object 2 in images used for analysis. * percent of (object 1) in (object 2): percent of the given segmented object 1 overlapping with the given segmented object 2 in images used for analysis. * avg percent of simulated (object 1) in (object 2): percentage of given object 1s that would overlap with object 2 by random chance given their respective abundance, sizes, and mitochondrial area. Generated using custom `object_intersection_sim.py` python file in `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/run_object_simulation/`. ##### Figure 03 Images and quantification showing that mitochondrial translation hubs are dynamic. * Figure 03A: * 15minPulse_HPG-647_TOM20-488_dsDNA-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 15minChase_HPG-647_TOM20-488_dsDNA-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and a 15 minute chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 30minChase_HPG-647_TOM20-488_dsDNA-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and a 30 minute chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 1hrChase_HPG-647_TOM20-488_dsDNA-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and a 1 hour chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * Figure 03B-C-D: * 03B-C-D Images Analyzed: All images used in the quantification of mitochondrial HPG intensity via variable chase times. * Chase_Avg_HPG_Area_Data.csv: Data representing the average area of segmented HPG objects in variable HPG chase images generated using `New HPG Segmentation Pipeline 1000 HPG threshold.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * 15 min Pulse (1000 Thresh): Average area, in um^2, of segmented HPG objects in the 15 minute pulse (5 minute chase) condition. * 15 min Chase (1000 Thresh): Average area, in um^2, of segmented HPG objects in the 15 minute chase condition. * 30 min Chase (1000 Thresh): Average area, in um^2, of segmented HPG objects in the 30 minute chase condition. * 1hr Chase (1000 Thresh): Average area, in um^2, of segmented HPG objects in the 1 hour chase condition. * Chase_Avg_HPG_Density_Data.csv: Data representing the average density of segmented HPG objects per mitochondrial area in variable HPG chase images generated using `New HPG Segmentation Pipeline 1000 HPG threshold.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * 15 min Pulse (1000 Thresh): Average density per mitochondrial area of segmented HPG objects in the 15 minute pulse (5 minute chase) condition. * 15 min Chase (1000 Thresh): Average density per mitochondrial area of segmented HPG objects in the 15 minute chase condition. * 30 min Chase (1000 Thresh): Average density per mitochondrial area of segmented HPG objects in the 30 minute chase condition. * 1hr Chase (1000 Thresh): Average density per mitochondrial area of segmented HPG objects in the 1 hour chase condition. * Chase_Avg_HPG_Intensity_Data.csv: Data representing the average intensity of segmented HPG objects in variable HPG chase images generated using `New HPG Segmentation Pipeline 1000 HPG threshold.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * 15 min Pulse (1000 Thresh): Average intensity of segmented HPG objects in the 15 minute pulse (5 minute chase) condition. * 15 min Chase (1000 Thresh): Average intensity of segmented HPG objects in the 15 minute chase condition. * 30 min Chase (1000 Thresh): Average intensity of segmented HPG objects in the 30 minute chase condition. * 1hr Chase (1000 Thresh): Average intensity of segmented HPG objects in the 1 hour chase condition. * Figure 03E: * 15minPulse_HPG-647_TOM20-488_dsDNA-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 30minPulse_HPG-647_TOM20-488_dsDNA-405-05-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 30 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * 1hrPulse_HPG-647_TOM20-488_dsDNA-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 1 hour pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * Chloro_HPG-647_TOM20-488_dsDNA-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media first treated with 50 ug/uL chloramphenicol for 20 minutes and then visualized using a click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * Figure 03F-G-H: * 0F-G-H Images Analyzed: All images used in the quantification of mitochondrial HPG intensity via variable pulse times. * Pulse_Avg_HPG_Area_Data.csv: Data representing the average area of segmented HPG objects in variable HPG pulse images generated using `New HPG Segmentation Pipeline 2.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * 15 min Pulse: Average area, in um^2, of segmented HPG objects in the 15 minute pulse condition. * 30 min Pulse: Average area, in um^2, of segmented HPG objects in the 30 minute pulse condition. * 1 hr Pulse: Average area, in um^2, of segmented HPG objects in the 1 hour pulse condition. * Chloro Control: Average area, in um^2, of segmented HPG objects in the 15 minute pulse condition first treated with 50 ug/uL chloramphenicol for 20 minutes. * Pulse_Avg_HPG_Density_Data.csv: Data representing the average density of segmented HPG objects per mitochondrial area in variable HPG pulse images generated using `New HPG Segmentation Pipeline 2.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * 15 min Pulse: Average density per mitochondrial area of segmented HPG objects in the 15 minute pulse condition. * 30 min Pulse: Average density per mitochondrial area of segmented HPG objects in the 30 minute pulse condition. * 1 hr Pulse: Average density per mitochondrial area of segmented HPG objects in the 1 hour pulse condition. * Chloro Control: Average density per mitochondrial area of segmented HPG objects in the 15 minute pulse condition first treated with 50 ug/uL chloramphenicol for 20 minutes. * Pulse_Avg_HPG_Intensity_Data.csv: Data representing the average intensity of segmented HPG objects in variable HPG pulse images generated using `New HPG Segmentation Pipeline 2.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * 15 min Pulse: Average intensity of segmented HPG objects in the 15 minute pulse condition. * 30 min Pulse: Average intensity of segmented HPG objects in the 30 minute pulse condition. * 1 hr Pulse: Average intensity of segmented HPG objects in the 1 hour pulse condition. * Chloro Control: Average intensity of segmented HPG objects in the 15 minute pulse condition first treated with 50 ug/uL chloramphenicol for 20 minutes. * Figure 03I: * RNR2-647_DRP1K38A-mCherry_HPG-488_TOM20-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cell not expressing mCherry-DRP1K38A (c2) and immunolabeled with antibodies against TOM20 (c4), RNA-FISH against mitoribosomal RNA RNR2 (c1), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-488 to show mitochondrial translation (c3). * RNR2-647_DRP1K38A-mCherry_HPG-488_TOM20-405-12-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cell expressing mCherry-DRP1K38A (c2) and immunolabeled with antibodies against TOM20 (c4), RNA-FISH against mitoribosomal RNA RNR2 (c1), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-488 to show mitochondrial translation (c3). * Figure 03J-K-L-M: * 03J-K-L-M Images Analyzed: All images used in the quantification of HPG and RNR2 in cells expressing DRP1K38A. * DRP1K38A_RNR2_HPG_Quantification.csv: Data for quantifying the changes in RNR2 and HPG in cells expressing DRP1K38A generated using `RNR2 HPG DRP1K38A Pipeline v4.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DRP1K38A avg RNR2 area: Average area of segmented RNR2 objects in cells expressing DRP1K38A. * Control avg RNR2 area: Average area of segmented RNR2 objects in cells not expressing DRP1K38A. * DRP1K38A avg HPG area: Average area of segmented HPG objects in cells expressing DRP1K38A. * Control avg HPG area: Average area of segmented HPG objects in cells not expressing DRP1K38A. * DRP1K38A Avg Mito Pixel Int C1: Average channel 1 intensity of segmented mitochondrial objects in cells expressing DRP1K38A. * DRP1K38A Avg Mito Pixel Int C3:Average channel 3 intensity of segmented mitochondrial objects in cells expressing DRP1K38A. * Control Avg Mito Pixel Int C1: Average channel 1 intensity of segmented mitochondrial objects in cells not expressing DRP1K38A. * Control Avg Mito Pixel Int C3:Average channel 3 intensity of segmented mitochondrial objects in cells not expressing DRP1K38A. ##### Figure 04 Images and quantification showing that mtRNAs remodel into dsRNA-rich mesoscale bodies during stress. * Figure 04B: * SUV3KO_D4_J2-647_TOM20-488-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c1) and TOM20 (c2). * SUV3KO_D4_J2-647_TOM20-488-16-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c1) and TOM20 (c2). * SUV3KO_D7_J2-647_TOM20-488-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c1) and TOM20 (c2). * SUV3KO_D7_J2-647_TOM20-488-16-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c1) and TOM20 (c2). * Figure 04C: * SUV3KO_D7_RNR2-647_TOM20-488_J2-405-03-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), TOM20 (c2), and RNA-FISH against mitoribosomal RNA RNR2 (c1). Corresponds to example 3 or third_ROI.roi. * SUV3KO_D7_RNR2-647_TOM20-488_J2-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), TOM20 (c2), and RNA-FISH against mitoribosomal RNA RNR2 (c1). Corresponds to example 1 or first_ROI.roi. * SUV3KO_D7_RNR2-647_TOM20-488_J2-405-12-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), TOM20 (c2), and RNA-FISH against mitoribosomal RNA RNR2 (c1). Corresponds to example 2 or second_ROI.roi. ##### Figure 05 Images and quantification showing that mitochondrial stress body formation coincides with attenuation of mitoribosome translation. * Figure 05A: * 1W-SUV3KO_RNR2-647_L-HPG-488_dsDNA-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitoribosomal RNA RNR2 (c1), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-488 to show mitochondrial translation (c2). * 1W-SUV3KO_RNR2-647_L-HPG-488_dsDNA-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitoribosomal RNA RNR2 (c1), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-488 to show mitochondrial translation (c2). * Figure 05B: * 05E Images Analyzed: All images used in the quantification of HPG in cells transfected and un-transfected for guide RNA-CRISPR complexes against SUV3. * SUV3_HPG_Data.csv: Data for quantifying the changes in HPG in cells either transfected or un-transfected for guide RNA-CRISPR complexes against SUV3 generated using `SUV3 KO HPG Mito Only Analysis.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * SUV3KO KO Mitos: Average HPG fluorescence intensity in segmented mitochondria in cells transfected for guide RNA-CRISPR complexes against SUV3. * SUV3KO Ctr Mitos: Average HPG fluorescence intensity in segmented mitochondria in cells un-transfected for guide RNA-CRISPR complexes against SUV3. * SUV3KO Chloro KO Mitos: Average HPG fluorescence intensity in segmented mitochondria in cells transfected for guide RNA-CRISPR complexes against SUV3 treated with 50 ug/uL chloramphenicol for 20 minutes. * SUV3KO Chloro Ctr Mitos: Average HPG fluorescence intensity in segmented mitochondria in cells un-transfected for guide RNA-CRISPR complexes against SUV3 treated with 50 ug/uL chloramphenicol for 20 minutes. ##### Figure 06 Images and quantification showing that mtRNA remodeling in stress is downstream of active translation. * Figure 06A: * 06A Images Analyzed: All images used in the quantification of mitoribosomal RNA RNR2 in cells transfected for guide RNA-CRISPR complexes against SUV3 after either treatment with 0.1% DMSO or 50 ug/uL chloramphenicol. * CAP_Pretreatment_SUV3_Quantification.csv: Data for quantifying the changes in RNR2 segmented object size and intensity in cells transfected for guide RNA-CRISPR complexes against SUV3 first treated with either 0.1% DMSO or 50 ug/uL chloramphenicol generated using `SUV3KO RNR2 Pretreatment Pipeline v3.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DMSOpreV2_avg size of rnr2 obj: Average size of segmented RNR2 objects in cells pretreated with 0.1% DMSO and 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * ChloropreV2_avg size of rnr2 obj: Average size of segmented RNR2 objects in cells pretreated with 50 ug/uL chloramphenicol and 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * DMSOpreV2_avg rnr2 intensity in mito: Average channel 1 (RNR2) intensity in segmented mitochondrial objects in cells pretreated with 0.1% DMSO and 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * ChloropreV2_avg rnr2 intensity in mito: Average channel 1 (RNR2) intensity in segmented mitochondrial objects in cells pretreated with 50 ug/uL chloramphenicol and 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * Figure 06B: * 06B Images Analyzed: All images used in the quantification of mitoribosomal RNA RNR2 in cells transfected for guide RNA-CRISPR complexes against SUV3 for 7 days and then treated with 0.1% DMSO or 50 ug/uL chloramphenicol. * CAP_Posttreatment_SUV3_Quantification.csv: Data for quantifying the changes in RNR2 segmented object size and intensity in cells transfected for guide RNA-CRISPR complexes against SUV3 for 7 days and then treated with either 0.1% DMSO or 50 ug/uL chloramphenicol generated using `SUV3KO RNR2 Posttreatment Pipeline v3.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DMSOpostV2_avg size of rnr2 obj: Average size of segmented RNR2 objects in cells treated with 0.1% DMSO after 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * ChloropostV2_avg size of rnr2 obj: Average size of segmented RNR2 objects in cells treated with 50 ug/uL chloramphenicol after 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * DMSOpostV2_avg rnr2 intensity in mito: Average channel 1 (RNR2) intensity in segmented mitochondrial objects in cells treated with 0.1% DMSO after 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * ChloropostV2_avg rnr2 intensity in mito: Average channel 1 (RNR2) intensity in segmented mitochondrial objects in cells treated with 50 ug/uL chloramphenicol after 7 days post transfection with guide RNA-CRISPR complexes against the gene SUV3. * Figure 06C: * CAP_pre_SUV3KO_d7_RNR2-647_J2-488_TOM20-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c2), TOM20 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1), treated with 50 ug/uL chloramphenicol prior to transfection. * DMSO_pre_SUV3KO_d7_RNR2-647_J2-488_TOM20-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c2), TOM20 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1), treated with 0.1% DMSO prior to transfection. * Figure 06D: * SUV3KO_D4_RNR2-647_J2-488_GRSF1-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c2), GRSF1 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * SUV3KO_D4_RNR2-647_J2-488_GRSF1-405-11-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c2), GRSF1 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * Figure 06E: * SUV3_D4_RNR2-647_HPG-488_J2-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), RNA-FISH against mitoribosomal RNA RNR2 (c1), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-488 to show mitochondrial translation (c2). * SUV3_D4_RNR2-647_HPG-488_J2-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), RNA-FISH against mitoribosomal RNA RNR2 (c1), and 15 minute pulse of 50 uM HPG and a 5 minute chase of HPG free media visualized using a click reaction with AlexaFlour-488 to show mitochondrial translation (c2). #### *Supplemental Figure Raw Data* All images and data used to generate the supplemental figures. Sub-directories are separated by figure number and further separated by their corresponding panel of that figure. The image shown in the figure as well as its metadata are included for each panel and the images that went into the given analysis are located in the corresponding folder named Images Analyzed. Raw csv files for the shown quantification are given as well with variable names corresponding to the respective figure and condition ``` |---Figure S1 |---S1B |---S1C |---RNR2 |---tRNAs |---ND4 |---S1D |---S1D Images Analyzed |---DMSO |---IMT1B |---RNaseA |---Figure S2 |---S2A |---S2B |---S2C |---S2D |---S2E |---S2F |---S2G |---Figure S3 |---S3A-B |---S3C-D-E |---S3C-D-E Images Analyzed |---S3F |---S3H |---S3H Images Analyzed |---Figure S4 |---Figure S5 |---S5A |---S5A Images Analyzed |---S5B |---S5C |---S5C Images Analyzed |---S5D |---S5E |---S5E Images Analyzed |---S5F |---S5G |---S5H |---S5I |---S5I Images Analyzed |---Figure S6 |---S6A |---S6B |---Control |---WT |---CatDead |---S6C |---S6C Images Analyzed |---output csvs |---Figure S7 |---Figure S8 |---S8A |---S8B |---S8D-E |---05B-C Movies Analyzed |---S8C |---Figure S9 |---S9A |---S9B |---S9B Images Analyzed |---S9C |---S9D |---S9E ``` ##### Figure S1 Images and quantification showing that specifically visualizing mitochondrial DNA encoded RNAs via RNA-FISH. * S1B * Unprocessed-647_COX3-594_TOM20-488_dsDNA-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), dsDNA (c4), and RNA-FISH against unprocessed mitochondrial RNA transcripts (c1) and mitochondrial mRNA COX3 (c2). * S1C * ND4: * DMSO96hr_ND4-647_TOM20-488_dsDNA-405-05-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial mRNA ND4 (c1) treated with 0.1% DMSO for 96 hours prior to fixation. * IMT1B96hr_ND4-647_TOM20-488_dsDNA-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial mRNA ND4 (c1) treated with 10uM IMT1B for 96 hours prior to fixation. * RNaseA_ND4-647_TOM20-488_dsDNA-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial mRNA ND4 (c1) treated with 100 ug/mL RNase A for 1 hour at RT prior to RNA-FISH. * RNR2: * DMSO96hr_RNR2-647_TOM20-488_dsDNA-405-01-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1) treated with 0.1% DMSO for 96 hours prior to fixation. * IMT1B96hr_RNR2-647_TOM20-488_dsDNA-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1) treated with 10uM IMT1B for 96 hours prior to fixation. * RNaseA_RNR2-647_TOM20-488_dsDNA-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1) treated with 100 ug/mL RNase A for 1 hour at RT prior to RNA-FISH. * tRNAs: * DMSO96hr_tRNAs-647_TOM20-488_dsDNA-405-03-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial tRNAs (c1) treated with 0.1% DMSO for 96 hours prior to fixation. * IMT1B96hr_tRNAs-647_TOM20-488_dsDNA-405-01-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial tRNAs (c1) treated with 10uM IMT1B for 96 hours prior to fixation. * RNaseA_tRNAs-647_TOM20-488_dsDNA-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial tRNAs (c1) treated with 100 ug/mL RNase A for 1 hour at RT prior to RNA-FISH. * S1D * S1D Images Analyzed: All images used in the quantification of mitoribosomal RNA RNR2, mitochondrial mRNA ND4, and mitochondrial tRNAs intensity in segmented mitochondrial objects treated with either DMSO, IMT1B, or RNase A. mtRNA Glut Fix Pipeline * ND4_Quantification.csv: Data for quantifying the changes in mitochondrial mRNA ND4 intensity in segmented mitochondrial objects in each condition generated using `mtRNA Glut Fix Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DMSO avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 0.1% DMSO for 96 hours. * IMT1B avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 10uM IMT1B for 96 hours. * RNaseA avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 100 ug/mL RNase A for 1 hour at RT prior to RNA-FISH. * RNR2_Quantification.csv: Data for quantifying the changes in mitoribosomal RNA RNR2 intensity in segmented mitochondrial objects in each condition generated using `mtRNA Glut Fix Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DMSO avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 0.1% DMSO for 96 hours. * IMT1B avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 10uM IMT1B for 96 hours. * RNaseA avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 100 ug/mL RNase A for 1 hour at RT prior to RNA-FISH. * tRNAs_Quantification.csv: Data for quantifying the changes in mitochondrial tRNAs intensity in segmented mitochondrial objects in each condition generated using `mtRNA Glut Fix Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DMSO avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 0.1% DMSO for 96 hours. * IMT1B avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 10uM IMT1B for 96 hours. * RNaseA avg channel 1 int: Avg channel 1 intensity in segmented mitochondrial objects pretreated with 100 ug/mL RNase A for 1 hour at RT prior to RNA-FISH. ##### Figure S2 Images and quantification showing that RNA-FISH of other mitochondrial DNA encoded RNAs. * S2A: * ATP6-647_TOM20-488_dsDNA-405-01-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial mRNA ATP6 (c1). * LinescanData.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S2B: * control_COX1-647_TOM20-488_dsDNA-405-08-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and RNA-FISH against mitochondrial mRNA COX1 (c1). * LinescanData.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S2C: * RNR1-647_RNR2-560_TOM20-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), and RNA-FISH against mitoribosomal RNAs RNR1 (c1), and RNR2 (c2). * LinescanData.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S2D: * ATP6-647_HPG-488_TOM20-405-03-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitoribosomal mRNA ND4 (c1), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c2). * linescandata.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S2E: * COX1-647_HPG-488_TOM20-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitoribosomal mRNA COX1 (c1), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c2). * LinescanData.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S2F: * COX1-647_ND4-560_HPG-488_TOM20-405-05-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c4), RNA-FISH against mitoribosomal mRNA COX1 (c1) and ND4 (c2), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c3). * linescan_data.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S2G: * ND2-647_ND4-560_HPG-488_TOM20-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c4), RNA-FISH against mitoribosomal mRNA ND2 (c1) and ND4 (c2), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-488 to show mitochondrial translation (c3). * LinescanData.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. ##### Figure S3 Images and quantification showing that Labeling nascent mitochondrial transcription with the metabolic label EU and iterative linescan analysis. * S3A-B: * IMR90_1hrEU_GRSF1_TOM20_1CLICK-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against GRSF1 (c2), TOM20 (c3), and nascent mtRNA labeling using 500uM EU for 1 hour and following click reaction with AlexaFlour-647 (c1). * LinescanData.csv: Data for fluorescent intensities for each imaging channel listed above across a representative linescan distance in microns. * S3C-D-E: * S3C-D-E Images Analyzed: All images used in the quantification of nascent mtRNA overlap with GRSF1. * EU_Quantification.csv: Data for quantifying the overlap between EU and GRSF1 objects generated from `EU GRSF1 Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * number of EU per mito area: number of segmented EU objects normalized to segmented mitochondrial area. * number of GRSF1 per mito area: number of segmented GRSF1 objects normalized to segmented mitochondrial area. * percent of EU in GRSF1: percent of segmented EU objects overlapping with segmented GRSF1 objects. * percent of EU in GRSF1 above Sim: percent of segmented EU objects overlapping with segmented GRSF1 objects above what is expected by random chance given their respective abundance, sizes, and mitochondrial area. Generated using custom `object_intersection_sim.py` python file in `Code used in Paper/Begeman_et_al_Python_Code/ObjectIntersection/run_object_simulation/`. * S3F: * IMR90_1hrEU_GRSF1_TOM20_1CLICK-01-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against GRSF1 (c2), TOM20 (c3), and nascent mtRNA labeling using 500uM EU for 1 hour and following click reaction with AlexaFlour-647 (c1). * imr90_grsf1_dna_mtrkr-06-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against GRSF1 (c2), dsDNA (c3), and mitochondria labeled with 100nM Mito tracker deep red for 30 minutes prior to fixation. * imr90_mtdr_dna_tfam-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TFAM (c2), dsDNA (c3), and mitochondria labeled with 100nM Mito tracker deep red for 30 minutes prior to fixation. * S3H: * S3H Images Analyzed: All images used in the analysis of EU or dsDNA interactions with GRSF1 and TFAM. * EU_GRSF1_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of EU (c1) and GRSF1 (c2). Data generated by first segmenting mitochondrial objects using `EU GRSF1 Pipeline.pipeline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * GRSF1_DNA_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of GRSF1 (c2) and dsDNA (c3). Data generated by first segmenting mitochondrial objects using `GRSF1 and DNA Pipeline v2.pipline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * TFAM_DNA_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of TFAM (c2) and dsDNA (c3). Data generated by first segmenting mitochondrial objects using `TFAM and DNA Pipeline.pipeline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis ##### Figure S4 Images and quantification showing that mitochondrial RNA labeling with EU coupled to RNA-FISH. * BEST_EU-647_ND4-570_TOM20-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitochondrial mRNA ND4 (c2), and nascent mtRNA labeling using 500uM EU for 1 hour and following click reaction with AlexaFlour-647 (c1). * BEST_EU-647_RNR2-570_TOM20-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), RNA-FISH against mitoribosomal RNA RNR2 (c2), and nascent mtRNA labeling using 500uM EU for 1 hour and following click reaction with AlexaFlour-647 (c1). * linescan_data_nd4.csv: CSV file of fluorescent intensities for each imaging channel listed above for ND4 across a representative linescan distance. * linescan_data_rnr2.csv: CSV file of fluorescent intensities for each imaging channel listed above for RNR2 across a representative linescan distance. ##### Figure S5 Images and quantification showing that extended RNA-FISH and HPG data. * S5A: * S5A Images Analyzed: All images used in the quantification of the overlap between segmented ND4 and MRPL23 objects. * ND4_MRPL23_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of ND4 (c1) and MRPL23 (c2). Data generated by first segmenting mitochondrial objects using `ND4 (Blob) and MRPL23 Pipeline.pipeline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * S5B: * RNR2-647_MRPL23-488_TOM20-405-05-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against MRPL23 (c2), TOM20 (c3), and RNA-FISH targeting mitoribosomal component RNR2 (c1). * S5C: * S5C Images Analyzed: All images used in the quantification of the overlap between segmented RNR2 and MRPL23 objects. * RNR2_MRPL23_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of RNR2 (c1) and MRPL23 (c2). Data generated by first segmenting mitochondrial objects using `RNR2 and MRPL23 Pipeline.pipeline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * S5D: * tRNAs-647_MRPL23-488_TOM20-405-09-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against MRPL23 (c2), TOM20 (c3), and RNA-FISH targeting mitochondrial tRNAs (c1). * S5E: * S5E Images Analyzed: All images used in the quantification of the overlap between segmented mt-tRNAs and MRPL23 objects. * tRNAs_MRPL23_Linescan_Analysis_Data.csv: Data for iterative linescan analysis of mt-tRNAs (c1) and MRPL23 (c2). Data generated by first segmenting mitochondrial objects using `tRNAs and MRPL23 Pipeline.pipeline` located in `Code used in Paper/Begeman_et_al_Arivis_Code/` which were then used in Fiji macro `SkeletontoLineScan.imj.imj.py` located in `Code used in paper/Begeman_et_al_FIJI_Code/` to generate thousands of individual linescans of fluorescence intensity that were then used to generate average linescan using custom python code located at `Code used in Paper/Begeman_et_al_Python_Code/AvgLineScan/` with the following column labels. * Distance: distance in microns * Headers follow pattern: C(channel number used for peak calling) Channel (channel of fluorescent intensity) * Mean: mean fluorescence intensity * SD: standard deviation in fluorescence intensity * N: number of individual linescans that go into iterative analysis * S5F: * 15minHPG-647_GRSF1-488_TOM20-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), GRSF1 (c2), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * S5G: * 15minHPG-647_TFAM-488_TOM20-405-05-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c3), TFAM (c2), and 15 minute pulse of 50 uM HPG and following click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * S5H: * 1hrPulse_HPG-647_TOM20-488_dsDNA-405-07-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells immunolabeled with antibodies against TOM20 (c2), dsDNA (c3), and 1 hour pulse of 50 uM HPG and following click reaction with AlexaFlour-647 to show mitochondrial translation (c1). * S5I: * S5I Images Analyzed: All images used in the quantification of HPG and RNR2 in cells expressing DRP1K38A. * Histogram_values.csv: Data quantifying the changes in segmented mitochondrial length per area between DRP1K38A expressing cells and cells not expressing the protein generated using `RNR2 HPG DRP1K38A Pipeline v4.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * DRP1K38A mito length per area: The length of segmented mitochondrial objects over their areas in cells expressing DRP1K38A. * ctn mito length per area: The length of segmented mitochondrial objects over their areas in cells not expressing DRP1K38A. ##### Figure S6 Images and quantification showing that SUV3-HA expression and extended sgSUV3 data. * S6A: * SUV3HAtransfection_MTDR-647_GRSF1-488_HA-405-01-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells expressing HA-SUV3 and immunolabeled with antibodies against GRSF1 (c2), HA (c3), and mitochondria labeled using 100nM Mito tracker deep red 30 minutes prior to fixation. * LinescanData.csv: CSV file of fluorescent intensities for each imaging channel listed above across a representative linescan distance. * S6B: * CatDead: * CatDeadSUV3Rescue_RNR2-647_HA-488_TOM20-405-22-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection expressing a catalytically dead helicase mutant HA-SUV3 and immunolabeled with antibodies against HA (c2), TOM20 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * Control: * CatDeadSUV3Rescue_RNR2-647_HA-488_TOM20-405-28-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection not expressing a catalytically dead helicase mutant HA-SUV3 and immunolabeled with antibodies against HA (c2), TOM20 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * WT: * WTSUV3Rescue_RNR2-647_HA-488_TOM20-405-15-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection expressing HA-SUV3 and immunolabeled with antibodies against HA (c2), TOM20 (c3), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * S6C: * S6C Images Analyzed: All images used in the quantification of the rescue of CRISPR SUV3 KO by expressing WT and catalytically dead SUV3. * Rescue_Quantification.csv: Data for quantifying the percent of the mitochondrial network covered with SUV3 induced mitochondrial stress bodies as marked by mitoribosomal RNA RNR2 normalized to SUV3 KO cells not expressing a SUV3 protein generated using `SUV3 WT Rescue Pipeline First Bio.pipeline` and `SUV3 Cat Dead Rescue Pipeline.pipeline` located at `Code used in Paper/Begeman_et_al_Arivis_Code/` with the following column labels. * CatDead: percent of the mitochondrial network in cells expressing the catalytically dead SUV3 mutant covered with SUV3 induced mitochondrial stress bodies as marked by mitoribosomal RNA RNR2 normalized to SUV3 KO cells not expressing a SUV3 protein. * Control: percent of the mitochondrial network in cells expressing the catalytically dead SUV3 mutant covered with SUV3 induced mitochondrial stress bodies as marked by mitoribosomal RNA RNR2 normalized to SUV3 KO cells not expressing a SUV3 protein. * WT: percent of the mitochondrial network in cells expressing the catalytically dead SUV3 mutant covered with SUV3 induced mitochondrial stress bodies as marked by mitoribosomal RNA RNR2 normalized to SUV3 KO cells not expressing a SUV3 protein. ##### Figure S7 Images and quantification showing that messenger RNA localization in cells depleted of SUV3. * 1W-SUV3KO_ND4-647_TOM20-488_dsDNA-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against dsDNA (c3), TOM20 (c2), and RNA-FISH against mitochondrial mRNA ND4 (c1). * 1W-SUV3KO_ND4-647_TOM20-488_dsDNA-405-09-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against dsDNA (c3), TOM20 (c2), and RNA-FISH against mitochondrial mRNA ND4 (c1). ##### Figure S8 Images and quantification showing that live cell dynamics of mitochondrial stress bodies. * S8A: * SUV3KO-1w_mtkrdr-647_RNAselect-488-01-Airyscan Processing.czi: Representative image and image metadata for live IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection with mitochondrial RNA labeled using 5uM RNASelect for 1 hour and mitochondria labeled with 100nM Mito tracker deep red for 30 minutes prior to imaging. * SUV3KO-1w_mtkrdr-647_RNAselect-488-06-Airyscan Processing.czi: Representative image and image metadata for live IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection with mitochondrial RNA labeled using 5uM RNASelect for 1 hour and mitochondria labeled with 100nM Mito tracker deep red for 30 minutes prior to imaging. * S8B: * SUV3KO-1w_TMRE-561-09-Airyscan Processing.czi: Representative image and image metadata for live IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection with mitochondria labeled with 500nM TMRE for 30 minutes prior to imaging. * SUV3KO-1w_TMRE-561-15-Airyscan Processing.czi: Representative image and image metadata for live IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection with mitochondria labeled with 500nM TMRE for 30 minutes prior to imaging. * S8C: * FRAPTEST_SUV3KO-1w_mtkrdr-647-06-Airyscan Processing.czi: Representative movie and movie metadata for live IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection with mitochondrial RNA labeled using 5uM RNASelect for 1 hour and mitochondria labeled with 100nM Mito tracker deep red for 30 minutes prior to imaging. Bleached region located `BleachCallout.roi` and non bleached region located `NonBleachCallout.roi`. * S8D-E: * 05B-C Movies Analyzed: All movies used in the quantification of fluorescence recovery of mitochondrial stress bodies labeled by RNASelect in SUV3 KO cells. * Recovery_Over_Time_Data.csv: Data for the recovery of fluorescence signal in the RNASelect channel of mitochondrial stress bodies in regions bleached and nonbleached with the following columns. * Time: time post in seconds. * Bleach Max: Maximum fluorescence intensity of RNASelect in bleached region. * Bleach Avg: Average fluorescence intensity of RNASelect in bleached region. * Nonbleach Max: Maximum fluorescence intensity of RNASelect in nonbleached region. * Nonbleach Average: Average fluorescence intensity of RNASelect in nonbleached region. * Bleach Adj. Bleach Max: Maximum fluorescence intensity of RNASelect in bleached region normalized for imaging bleaching overtime. * Bleach Adj. Bleach Avg: Average fluorescence intensity of RNASelect in bleached region normalized for imaging bleaching overtime. * Bleach Adj. Nonbleach Max: Maximum fluorescence intensity of RNASelect in nonbleached region normalized for imaging bleaching overtime. * Bleach Adj. Nonbleach Average: Average fluorescence intensity of RNASelect in nonbleached region normalized for imaging bleaching overtime. * Recovery_Percentage_Data.csv: Data for percentage recovery of 7 examples of mitochondrial stress bodies with the following column labels. * Start: Percentage of total signal before bleaching normalized to start. * End: Percentage of total signal after recovery from bleaching normalized to start. * Movie {number}: Movie being analyzed. ##### Figure S9 Images and quantification showing that GRSF1 and PRORP, but not LRPPRC, are enriched in MSBs. * S9A: * 1W-SUV3KO_RNR2-647_GRSF1-488_TOM20-405-03-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against TOM20 (c3), GRSF1 (c2), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * 1W-SUV3KO_RNR2-647_GRSF1-488_TOM20-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 7 days post transfection immunolabeled with antibodies against TOM20 (c3), GRSF1 (c2), and RNA-FISH against mitoribosomal RNA RNR2 (c1). * S9B: * S9B Images Analyzed: All images used in the quantification of GRSF1 size in control and SUV3 KO cells. * Quantification.csv: Data quantifying the size of GRSF1 segmented objects in cells transfected and un-transfected for guide RNA-CRISPR complexes against SUV3 7 days post transfection with the following labels: * SUV3 Surface Area (µm²): size of segmented GRSF1 objects in SUV3 KO cells. * WT Surface Area (µm²): size of segmented GRSF1 objects in control cells. * S9C: * NTRKO1W_Mtdr-647_LRPPRC-488_J2-405-04-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against scramble RNA 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), LRPPRC (c2), mitochondria labeled with 100nM Mito tracker deep red (c1) for 30 minutes prior to fixation. * SUV3KO1W_Mtdr-647_LRPPRC-488_J2-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against SUV3 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), LRPPRC (c2), mitochondria labeled with 100nM Mito tracker deep red (c1) for 30 minutes prior to fixation. * S9D: * NTRKO1W_Mtdr-647_PRORP-488_J2-405-02-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against scramble RNA 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), PRORP (c2), mitochondria labeled with 100nM Mito tracker deep red (c1) for 30 minutes prior to fixation. * SUV3KO1W_Mtdr-647_PRORP-488_J2-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells transfected for guide RNA-CRISPR complexes against SUV3 7 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), PRORP (c2), mitochondria labeled with 100nM Mito tracker deep red (c1) for 30 minutes prior to fixation. * S9E: * SUV3KO_D4_RNR2-647_TOM20-488_J2-405-10-Airyscan Processing.czi: Representative image and image metadata of fixed IMR90 cells un-transfected for guide RNA-CRISPR complexes against the SUV3 gene 4 days post transfection immunolabeled with antibodies against J2 (dsRNA) (c3), TOM20 (c2), and RNA-FISH against mitoribosomal RNA RNR2 (c1). ## Sharing/Access Information All data and code can also be found on the Lewis Lab Github: [https://github.com/TheLewisLab](https://github.com/TheLewisLab)