Data from: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

Begeman, Adam 1 ; Smolka, John1; Shami, Ahmad1; Waingankar, Tejashree1; Lewis, Samantha 1

Published May 21, 2025 on Dryad. https://doi.org/10.5061/dryad.rxwdbrvkt

Data files

May 21, 2025 version files 79.99 GB

Begemanetal_Sci_Adv_2025_Raw_Data.zip
79.99 GB
README.md
81.89 KB

Abstract

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.

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

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