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Figure Lengend Snippet: a, dsRNA-seq experimental approach. b, dsRNA-seq reads across the mitochondrial genome spanning protein coding region in untreated HeLa cells. H-strand genes are shown as blue bars and L-strand as red bars. Short tRNA genes are denoted with T as the first letter. Data are representative of two experiments. c, Immunostaining of dsRNA in HeLa cells with anti-dsRNA (J2) antibody. Mitochondria and nuclei are stained with MitoTracker Deep Red and Hoechst, respectively. Scale bars, 10 μm. Graphs quantify co-localization of dsRNA foci with mitochondria. Data are mean ± s.d. from 29 cells. d, Anti-dsRNA (J2) staining in HeLa cells depleted for PNPase or SUV3 by siRNA as in c. Different imaging settings were applied in panel c and d so that the J2 intensity of control cells varies. e, Western blot showing PNPase or SUV3 depletion. Blots are representative of four experiments. f, Quantification of dsRNA levels in PNPase- or SUV3-depleted cells. Data are mean ± s.d. from four experiments. For gel source data, see Supplementary Fig. 1.

Article Snippet: The following antibodies were obtained commercially: mouse anti-ADAR1 mAb (sc-73408, Santa Cruz), rabbit anti-PNPT1 (ab96176, Abcam; sc-49315, Santa Cruz), mouse anti-dsRNA mAb J2 (10010500, Scions), anti-DNA (61014, Progen), rabbit anti-SUV3 (A303-055A, Bethyl Laboratories) 11 , mouse anti-RIG-I mAb (Alme-1) (AG-20B-0009, AdipoGen), rabbit anti-COX IV (3E11, Cell Signaling), rabbit anti-cytochrome c (NB100-91732, Novus Biologicals), rabbit anti-calnexin (2433, Cell Signaling), mouse anti-lamin A/C (4C11, Cell Signaling), donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 (A-21202, Lifetech), goat anti-mouse IgM conjugated with Alexa Fluor 555 (A-21426, Thermo Fisher Scientific), normal mouse IgG2a (sc-3878, Santa Cruz), mouse anti-MDA5 (in house from J.R.), rabbit anti-MAVS (ALX-210-929-C100, Enzo Life Sciences), rabbit anti-Bax (2772T, Cell Signaling), rabbit anti-Bak (6947T, Cell Signaling), rabbit anti-OXA1L (HPA003531, Sigma), mouse anti-α-Tubulin (T5168, Sigma), mouse anti-actin (ab8226, Abcam; A5441, Sigma), rabbit anti-Flag (PA1-984B, Thermo Fisher Scientific), HRP secondary anti-mouse (ab6728, Abcam; A9044, Sigma) 11 , HRP secondary anti-rabbit (A0545, Sigma; ab6721, Abcam) 11 , goat anti-mouse IgG (20 nm gold) preadsorbed (ab27242, Abcam).

Techniques: Immunostaining, Staining, Imaging, Western Blot

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Figure Lengend Snippet: a, RT–qPCR analysis of IFNB1 expression in HeLa cells transfected with indicated siRNAs. Data are mean ± s.d. from three independent experiments. b, RT–qPCR analysis of Ifit1 expression in Ddx58+/− (control, RIG-I+), Ddx58−/− (RIG-I−) and Ifih1−/− (MDA5−) immortalized MEFs transfected with mtRNA, RIG-I (ppp-IVT-RNA99nt) or MDA5 (CIP-EMCV RNA) specific agonists. Data are the mean from two independent experiments. Values are plotted on a logarithmic scale. c, Left, RT–qPCR analysis of IFNB1 mRNA in HeLa cells treated with the indicated siRNAs. Data are mean from two independent experiments. Right, western blot of siRNA depletion efficiency. Blots are representative of two experiments. For gel source data, see Supplementary Fig. 1. d, Model of mtdsRNA suppression by the RNA degradosome. Loss of PNPase causes accumulation of mtdsRNA and release of mtdsRNA into the cytoplasm in a Bax–Bak dependent manner. PNPase restricts mtdsRNA in matrix (together with SUV3) and IMS. MDA5 acts as the primary mtdsRNA sensor transducing an interferon response through the MAVS signalling pathway.

Article Snippet: The following antibodies were obtained commercially: mouse anti-ADAR1 mAb (sc-73408, Santa Cruz), rabbit anti-PNPT1 (ab96176, Abcam; sc-49315, Santa Cruz), mouse anti-dsRNA mAb J2 (10010500, Scions), anti-DNA (61014, Progen), rabbit anti-SUV3 (A303-055A, Bethyl Laboratories) 11 , mouse anti-RIG-I mAb (Alme-1) (AG-20B-0009, AdipoGen), rabbit anti-COX IV (3E11, Cell Signaling), rabbit anti-cytochrome c (NB100-91732, Novus Biologicals), rabbit anti-calnexin (2433, Cell Signaling), mouse anti-lamin A/C (4C11, Cell Signaling), donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 (A-21202, Lifetech), goat anti-mouse IgM conjugated with Alexa Fluor 555 (A-21426, Thermo Fisher Scientific), normal mouse IgG2a (sc-3878, Santa Cruz), mouse anti-MDA5 (in house from J.R.), rabbit anti-MAVS (ALX-210-929-C100, Enzo Life Sciences), rabbit anti-Bax (2772T, Cell Signaling), rabbit anti-Bak (6947T, Cell Signaling), rabbit anti-OXA1L (HPA003531, Sigma), mouse anti-α-Tubulin (T5168, Sigma), mouse anti-actin (ab8226, Abcam; A5441, Sigma), rabbit anti-Flag (PA1-984B, Thermo Fisher Scientific), HRP secondary anti-mouse (ab6728, Abcam; A9044, Sigma) 11 , HRP secondary anti-rabbit (A0545, Sigma; ab6721, Abcam) 11 , goat anti-mouse IgG (20 nm gold) preadsorbed (ab27242, Abcam).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot

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Figure Lengend Snippet: Oligonucleotide primers and siRNAs used in the study

Article Snippet: The following antibodies were obtained commercially: mouse anti-ADAR1 mAb (sc-73408, Santa Cruz), rabbit anti-PNPT1 (ab96176, Abcam; sc-49315, Santa Cruz), mouse anti-dsRNA mAb J2 (10010500, Scions), anti-DNA (61014, Progen), rabbit anti-SUV3 (A303-055A, Bethyl Laboratories) 11 , mouse anti-RIG-I mAb (Alme-1) (AG-20B-0009, AdipoGen), rabbit anti-COX IV (3E11, Cell Signaling), rabbit anti-cytochrome c (NB100-91732, Novus Biologicals), rabbit anti-calnexin (2433, Cell Signaling), mouse anti-lamin A/C (4C11, Cell Signaling), donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 (A-21202, Lifetech), goat anti-mouse IgM conjugated with Alexa Fluor 555 (A-21426, Thermo Fisher Scientific), normal mouse IgG2a (sc-3878, Santa Cruz), mouse anti-MDA5 (in house from J.R.), rabbit anti-MAVS (ALX-210-929-C100, Enzo Life Sciences), rabbit anti-Bax (2772T, Cell Signaling), rabbit anti-Bak (6947T, Cell Signaling), rabbit anti-OXA1L (HPA003531, Sigma), mouse anti-α-Tubulin (T5168, Sigma), mouse anti-actin (ab8226, Abcam; A5441, Sigma), rabbit anti-Flag (PA1-984B, Thermo Fisher Scientific), HRP secondary anti-mouse (ab6728, Abcam; A9044, Sigma) 11 , HRP secondary anti-rabbit (A0545, Sigma; ab6721, Abcam) 11 , goat anti-mouse IgG (20 nm gold) preadsorbed (ab27242, Abcam).

Techniques: Sequencing, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *

doi: 10.1074/jbc.M113.536540

Figure Lengend Snippet: SUV3, PNPase, and mtPAP interact in vivo to form a large molecular mass complex when the ETC is disrupted. A and B, co-immunoprecipitation assays of SUV3 by immunoprecipitating mtPAP or PNPase, respectively, using whole cell lysate post 1 h of oligomycin A (Oligo) or Az and 2-DG treatments. C, control, culture media containing DMSO only. All proteins were visualized by immunoblotting. C, gel filtration (Superdex 200) fractionations of whole cell lysate post 1 h of Az and 2-DG treatments. Approximate molecular masses of gel filtration fractions were determined by calibrating the column with known size markers. SUV3, mtPAP, and PNPase were resolved by SDS-PAGE and visualized by immunoblotting. D, co-immunoprecipitation of mtPAP and PNPase by immunoprecipitating SUV3 with large molecular mass fractions (*). E and F, reciprocal co-immunoprecipitation (IP) of mtPAP and PNPase with large (*) and small (**) gel filtration fractions, respectively.

Article Snippet: Clarified cell lysate containing 300 μg of protein was incubated with either 1 μg of rabbit polyclonal antibody for SUV3, PNPase, or mtPAP (GeneTex, Irvine, CA), or rabbit IgG at 4 °C on a rotator for 2 h. Subsequently, 50 μl of bovine serum albumin (BSA)-blocked protein G-Sepharose beads (GeneTex, Irvine, CA) was added to the mixture and incubated at 4 °C on a rotator for an additional hour.

Techniques: In Vivo, Immunoprecipitation, Control, Western Blot, Filtration, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *

doi: 10.1074/jbc.M113.536540

Figure Lengend Snippet: SUV3 serves as a bridge for PNPase and mtPAP binding. A, in vitro binding assay. GST-mtPAP full-length (FL) or GST proteins were bound to glutathione-Sepharose 4B beads. Group C was preincubated with increasing amounts of His-SUV3 for 1 h. Subsequently, Groups A, B, and C were incubated with increasing amounts of His-PNPase, whereas Group D was incubated with increasing amounts of preformed SUV3·PNPase complex for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. B, in vitro binding assay. Increasing amounts of un-tagged SUV3 was preincubated with His-PNPase on Ni+ resin for 1 h. Subsequently, equal amounts of GST-mtPAP were added to the mixture and further incubated for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. C, size exclusion chromatography (Superdex 200) elution profiles of the purified recombinant SUV3·PNPase·mtPAP complex and the individual proteins. The molecular masses (M.M.) of the individual proteins were determined by analytical ultracentrifuge. The molecular mass of the SUV3·PNPase·mtPAP complex was approximated from its peak elution volume. D, SDS-PAGE followed by silver staining of the complex. E, schematic representation of the SUV3·PNPase·mtPAP complex.

Article Snippet: Clarified cell lysate containing 300 μg of protein was incubated with either 1 μg of rabbit polyclonal antibody for SUV3, PNPase, or mtPAP (GeneTex, Irvine, CA), or rabbit IgG at 4 °C on a rotator for 2 h. Subsequently, 50 μl of bovine serum albumin (BSA)-blocked protein G-Sepharose beads (GeneTex, Irvine, CA) was added to the mixture and incubated at 4 °C on a rotator for an additional hour.

Techniques: Binding Assay, In Vitro, Incubation, SDS Page, Western Blot, Size-exclusion Chromatography, Purification, Recombinant, Silver Staining

Journal: The Journal of Biological Chemistry

Article Title: Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *

doi: 10.1074/jbc.M113.536540

Figure Lengend Snippet: N-terminal region of SUV3 binds to the C-terminal region of mtPAP. A and C, schematics of SUV3 constructs used in the in vitro binding assays. B and D, in vitro binding assay. Top panels, immunoblotting of His-tagged mtPAP remained bound to GST-SUV3 fusion proteins on glutathione-Sepharose 4B beads after binding and washing. Bottom panels, protein quantifications of GST-SUV3 fusion proteins by Coomassie Blue staining. *, desired protein species. E and G, schematics of mtPAP constructs used for the in vitro binding assays. F and H, in vitro binding assay. Top panels: immunoblotting of His-tagged SUV3 remained bound to GST-mtPAP fusion proteins on-Sepharose 4B beads after binding and washing. Bottom panels, protein quantifications of GST-mtPAP fusion proteins by Coomassie Blue staining. FL, full-length.

Article Snippet: Clarified cell lysate containing 300 μg of protein was incubated with either 1 μg of rabbit polyclonal antibody for SUV3, PNPase, or mtPAP (GeneTex, Irvine, CA), or rabbit IgG at 4 °C on a rotator for 2 h. Subsequently, 50 μl of bovine serum albumin (BSA)-blocked protein G-Sepharose beads (GeneTex, Irvine, CA) was added to the mixture and incubated at 4 °C on a rotator for an additional hour.

Techniques: Construct, In Vitro, Binding Assay, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *

doi: 10.1074/jbc.M113.536540

Figure Lengend Snippet: SUV3 enhances the activity of mtPAP and PNPase by providing a robust ssRNA binding domain. A, summary table of the various SUV3 mutants pertinent to this study. B, protein quantification of GST-SUV3 WT, KRK, and FYK mutants by Coomassie Blue staining. 10 and 20 μl of protein-bound beads were loaded in lanes 2, 4, and 6, and lanes 1, 3, and 5, respectively. C, in vitro binding assay. Immunoblotting of His-tagged mtPAP remained bound to 20 μl of beads containing GST-SUV3 WT, KRK, and FYK after washing. D, helicase assay. Increasing amounts of SUV3 WT, KRK, and FYK mutants (5, 10, and 20 ng/μl) were incubated with the 3′ overhang helicase substrate at 37 °C for 30 min in the presence of 5 mm ATP. Lane HD, heat denatured; the reaction mixture containing the substrate was heated to 80 °C for 10 min to denature the duplex immediately before loading onto 15% native PAGE. Lane C, control, the size exclusion chromatography buffer containing no protein was added. E, G, and I, polyadenylation assays. mtPAP alone or mtPAP with SUV3 wild-type or the designated mutants were incubated with 30 nt ssRNA (3WRNA) at 37 °C in the presence of 1 mm ATP. In E, the amount of KRK used was twice that of SUV3 WT. In G and I, the respective concentrations of K213A and RII were the same as those of SUV3 WT. F, H, and J, respective quantifications for relative abundance of poly(A) tails that were longer than 50 nt in panels E, G, and I as indicated by dashed lines. AU, arbitrary unit.

Article Snippet: Clarified cell lysate containing 300 μg of protein was incubated with either 1 μg of rabbit polyclonal antibody for SUV3, PNPase, or mtPAP (GeneTex, Irvine, CA), or rabbit IgG at 4 °C on a rotator for 2 h. Subsequently, 50 μl of bovine serum albumin (BSA)-blocked protein G-Sepharose beads (GeneTex, Irvine, CA) was added to the mixture and incubated at 4 °C on a rotator for an additional hour.

Techniques: Activity Assay, Binding Assay, Staining, In Vitro, Western Blot, Helicase Assay, Incubation, Clear Native PAGE, Control, Size-exclusion Chromatography