a303 055a  (Bethyl)

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Schematic of mitochondrial transcript unwinding and degradation by SUV3 and PNPase complexes. ( B ) Representative images of cells immunolabeled for TOM20 (grayscale) and dsRNA (J2; blue/rainbow lookup table) at 4 days versus 7 days of SUV3 depletion. Scale bars, 5 μm. ( C ) Three representative images of RNA bodies in cells fixed and immunolabeled with antibodies against dsRNA (J2; blue), TOM20 (green), and RNA-FISH targeting RNR2 (magenta). Scale bars, 1 μm.

Article Snippet: To generate SUV3-HA and SUV3 G207V -HA, the human SUV3 cDNA was synthesized (Twist Bioscience) with or without the G207V amino acid substitution, including an HA tag appended to the C terminus of the sequence, and cloned into the pTwist cytomegalovirus-driven mammalian expression vector.

Techniques: Immunolabeling

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Representative image of control (top) and SUV3-depleted (bottom) cells pulse-labeled with 50 μM HPG for 15 min (green), fixed, and immunolabeled to detect TOM20 (blue) and RNA-FISH against RNR2 (magenta). Scale bars, 1 μm. ( B ) Average HPG fluorescence intensity in segmented mitochondria in the presence or absence of CAP (50 μg/ml) [(no CAP) control: n = 9 mitochondrial networks, sgSUV3: n = 6 mitochondrial networks; (CAP) control: n = 4 mitochondrial networks, sgSUV3: n = 4 mitochondrial networks]. [** P < 0.01, Kruskal-Wallis test (** P < 0.01), followed by Dunn’s multiple comparisons]. ( C ) Diagram of proposed impact of SUV3 depletion on dsRNA accumulation and single-stranded RNA reorganization into mitochondrial stress bodies.

Article Snippet: To generate SUV3-HA and SUV3 G207V -HA, the human SUV3 cDNA was synthesized (Twist Bioscience) with or without the G207V amino acid substitution, including an HA tag appended to the C terminus of the sequence, and cloned into the pTwist cytomegalovirus-driven mammalian expression vector.

Techniques: Control, Labeling, Immunolabeling, Fluorescence

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Average ROI area and fluorescence intensity of segmented RNR2-FISH signals in maximum intensity projections of imaged IMR90 cells when SUV3 depletion is induced in cells after incubation in CAP (50 μg/ml) (DMSO: n = 16, CAP: n = 12). (** P < 0.01, Mann-Whitney test). ( B ) Average size and fluorescence intensity of segmented RNR2-FISH signals in maximum intensity projections of IMR90 cells incubated in CAP (50 μg/ml), imaged 7 days after transfection with sgSUV3 RNP complexes (DMSO: n = 17, CAP: n = 18). (not significant, Mann-Whitney test). ( C ) Representative images of control (top) and 50 μM CAP-treated (bottom) cells 7 days after transfection with sgSUV3 RNP complexes, fixed, and immunolabeled to detect dsRNA (J2; blue), TOM20 (green), and RNA-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( D ) Representative image of control (top) and day 4 sgSUV3 (bottom) cells, fixed, and immunolabeled to detect dsRNA (J2; green), GRSF1 (blue), and RNR2-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( E ) Representative image of control (top) and day 4 sgSUV3 (bottom) cells, pulse-labeled with 50 μM HPG for 15 min (green), fixed, and immunolabeled to detect dsRNA (J2; blue) and RNA-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( F ) Proposed model for organization of mitochondrial gene expression into translational hubs and their remodeling during stress.

Article Snippet: To generate SUV3-HA and SUV3 G207V -HA, the human SUV3 cDNA was synthesized (Twist Bioscience) with or without the G207V amino acid substitution, including an HA tag appended to the C terminus of the sequence, and cloned into the pTwist cytomegalovirus-driven mammalian expression vector.

Techniques: Fluorescence, Incubation, MANN-WHITNEY, Transfection, Control, Immunolabeling, Labeling, Gene Expression

Journal: bioRxiv

Article Title: RNA triggers chronic stress during neuronal aging

doi: 10.1101/2025.08.04.668575

Figure Lengend Snippet: (A) Screen captures from the Agilent TapeStation Analysis Software v4.1.1 of RNA ScreenTapes loaded with samples from throughout the dsRNA purification protocol in HEK293T cells. Exogenous (exo.) dsRNA of a known size (∼150 bp) was tracked throughout the purification. (B) Volcano plot of the log 2 (Fold Change) of all MT-RNA transcripts identified by dsRNA-Seq in the input and immunoprecipitation (IP) samples of the Tdiff.2 transdifferentiated neuron line. (C) Same as (B) but for Tdiff.5 transdifferentiated neurons. (D) Same as (B) but for iPSC-diff.1 neurons. (E) Volcano plot of RNA-seq in transdifferentiated neurons (Tdiff.1/2/4/5) versus iPSC-derived neurons (n=3 replicates per line; N=4 lines per cohort). Orange data points denote nuclear-encoded mitochondrial genes whereas yellow data points denote MT-RNA transcripts. The MT-ND6, SUV3, and PNPT1 transcripts are indicated on the plot. (F) Boxplot of the Log 2 (Fold Change) of all transcripts (gray), nuclear-encoded mitochondrial transcripts (orange), and MT-RNA transcripts (yellow) in transdifferentiated neurons versus iPSC-derived neurons (n=3 replicates per line; N=4 lines per cohort). (G) Visualization of representative mitochondrial sequencing reads detected by dsRNA-Seq and PKR CLIP in the Tdiff.1 transdifferentiated neuron line. The shaded region denotes the reverse-transcribed MT-ND6 gene. The range of the y-axis values is denoted on the right side of the genomic track and was fixed for each pair of input and IP samples.

Article Snippet: The following antibodies were used for eCLIP: 1 μg Rabbit α-PKR (Cell Signaling Technology #12297S); 10 μg Rabbit α-PNPT1 (Bethyl #A303-917A); and 2 μg Rabbit α-SUV3 (Bethyl #A303-056A).

Techniques: Software, Purification, Immunoprecipitation, RNA Sequencing, Derivative Assay, Sequencing, Reverse Transcription

Journal: bioRxiv

Article Title: RNA triggers chronic stress during neuronal aging

doi: 10.1101/2025.08.04.668575

Figure Lengend Snippet: (A) Additional airyscan immunofluorescence-FISH images of the mitochondrial marker TOM70 (magenta) and MT-ND6 transcript (green) in Tdiff.1 neurons. Green arrowheads denote cytoplasmic MT-ND6 puncta; the white arrows denote the intensity profiles in the adjacent plot. Yellow Scale Bar = 2 μm. (B) Same as (A) but for the MT-RNR1 transcript in various transdifferentiated neuron lines (Tdiff.1/2/4). (C) Airyscan immunofluorescence images of the indicated mitochondrial proteins in Tdiff.1 neurons. Yellow Scale Bar = 2 μm. (D) Visualization of representative mitochondrial sequencing reads detected by PNPT1 eCLIP (n=2 replicates) in the indicated neuronal lines. The shaded region denotes the reverse-transcribed MT-ND6 gene. (E) Same as (D) but for SUV3 eCLIP.

Article Snippet: The following antibodies were used for eCLIP: 1 μg Rabbit α-PKR (Cell Signaling Technology #12297S); 10 μg Rabbit α-PNPT1 (Bethyl #A303-917A); and 2 μg Rabbit α-SUV3 (Bethyl #A303-056A).

Techniques: Immunofluorescence, Marker, Sequencing, Reverse Transcription

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Schematic of mitochondrial transcript unwinding and degradation by SUV3 and PNPase complexes. ( B ) Representative images of cells immunolabeled for TOM20 (grayscale) and dsRNA (J2; blue/rainbow lookup table) at 4 days versus 7 days of SUV3 depletion. Scale bars, 5 μm. ( C ) Three representative images of RNA bodies in cells fixed and immunolabeled with antibodies against dsRNA (J2; blue), TOM20 (green), and RNA-FISH targeting RNR2 (magenta). Scale bars, 1 μm.

Article Snippet: Plasmids encoding SUV3-HA, SUV3 G207V -HA, and mCherry-DRP1 K38A have been deposited to Addgene.

Techniques: Immunolabeling

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Representative image of control (top) and SUV3-depleted (bottom) cells pulse-labeled with 50 μM HPG for 15 min (green), fixed, and immunolabeled to detect TOM20 (blue) and RNA-FISH against RNR2 (magenta). Scale bars, 1 μm. ( B ) Average HPG fluorescence intensity in segmented mitochondria in the presence or absence of CAP (50 μg/ml) [(no CAP) control: n = 9 mitochondrial networks, sgSUV3: n = 6 mitochondrial networks; (CAP) control: n = 4 mitochondrial networks, sgSUV3: n = 4 mitochondrial networks]. [** P < 0.01, Kruskal-Wallis test (** P < 0.01), followed by Dunn’s multiple comparisons]. ( C ) Diagram of proposed impact of SUV3 depletion on dsRNA accumulation and single-stranded RNA reorganization into mitochondrial stress bodies.

Article Snippet: Plasmids encoding SUV3-HA, SUV3 G207V -HA, and mCherry-DRP1 K38A have been deposited to Addgene.

Techniques: Control, Labeling, Immunolabeling, Fluorescence

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Average ROI area and fluorescence intensity of segmented RNR2-FISH signals in maximum intensity projections of imaged IMR90 cells when SUV3 depletion is induced in cells after incubation in CAP (50 μg/ml) (DMSO: n = 16, CAP: n = 12). (** P < 0.01, Mann-Whitney test). ( B ) Average size and fluorescence intensity of segmented RNR2-FISH signals in maximum intensity projections of IMR90 cells incubated in CAP (50 μg/ml), imaged 7 days after transfection with sgSUV3 RNP complexes (DMSO: n = 17, CAP: n = 18). (not significant, Mann-Whitney test). ( C ) Representative images of control (top) and 50 μM CAP-treated (bottom) cells 7 days after transfection with sgSUV3 RNP complexes, fixed, and immunolabeled to detect dsRNA (J2; blue), TOM20 (green), and RNA-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( D ) Representative image of control (top) and day 4 sgSUV3 (bottom) cells, fixed, and immunolabeled to detect dsRNA (J2; green), GRSF1 (blue), and RNR2-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( E ) Representative image of control (top) and day 4 sgSUV3 (bottom) cells, pulse-labeled with 50 μM HPG for 15 min (green), fixed, and immunolabeled to detect dsRNA (J2; blue) and RNA-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( F ) Proposed model for organization of mitochondrial gene expression into translational hubs and their remodeling during stress.

Article Snippet: Plasmids encoding SUV3-HA, SUV3 G207V -HA, and mCherry-DRP1 K38A have been deposited to Addgene.

Techniques: Fluorescence, Incubation, MANN-WHITNEY, Transfection, Control, Immunolabeling, Labeling, Gene Expression