Journal: PLoS ONE

Article Title: How Do Human Cells React to the Absence of Mitochondrial DNA?

doi: 10.1371/journal.pone.0005713

Figure Lengend Snippet: (A) Sub-organellar localization of Elac2 by western-blot analysis on mitochondria isolated from HeLa cells. Specificity of antibody reactivity was proved by the CRM obtained using an in vitro translated (i.v.) Elac2. A CRM was present in mitochondria (M), in the mitochondrial matrix (Ma) and membranes (Mb), and barely detectable in the nucleus (N). ETHE1 antibody was used as a mitochondrial matrix control protein. (B) Proteinase K protection assay by western-blot analysis on freshly isolated mitochondria from HeLa cells. Lane 1, cell lysate; lane 2, intact mitochondria; lane 3, mitochondria treated with 100 µg/ml of proteinase K; lane 4, mitochondria treated with 0.5% of Triton-X100. (C) Mitochondrial in vivo import of HA-tagged human Elac2 N-terminal polypeptide expressed in Cos-7 cells. Lane 1: marker 46 kDa (MW). lane 2: immunoprecipitation of total radiolabeled proteins. lane 3: immunoprecipitation of total radiolabeled proteins after the addition of valinomycin that determines the elimination of the mitochondrial ΔΨ: as a consequence, mitochondrial import is abolished. lane 4: immunoprecipitation of in vitro translation product (i.v.). Arrows indicate the Elac2 precursor (Elac2) and the mature form (mElac2). (D) Confocal immunofluorescence on Cos7 cells. The green fluorescence corresponds to Elac2HA-specific immunoreactivity. The red fluorescence corresponds to immunoreactivity specific to mtSSB. The two immunofluorescence patterns overlap, as shown by confocal merge. Magnification: 40×.

Article Snippet: An affinity purified polyclonal antibody against ELAC2 was from ProteinTech, while the polyclonal antibody against Ethe1 was raised in rabbit by Neosystem .

Techniques: Western Blot, Isolation, In Vitro, Control, In Vivo, Marker, Immunoprecipitation, Immunofluorescence, Fluorescence

Journal: Orphanet Journal of Rare Diseases

Article Title: A homozygous splicing mutation in ELAC2 suggests phenotypic variability including intellectual disability with minimal cardiac involvement

doi: 10.1186/s13023-016-0526-8

Figure Lengend Snippet: Genome-wide genotyping and sequencing results. a ) Genome-wide homozygosity mapping analysis revealed one stretch of homozygous genotypes in all the investigated patients on chromosome17 (indicated by a red bar). b ) Whole-exome sequencing IGV of the affected children V2 and V10 showing a 1 bp substitution of a canonical splice site in all reads of ELAC2 exon 15 . Sequence of wild type gene on reverse strand (-) and exon annotation at the bottom. c ) Sanger sequencing verified that the c.1423 + 2 T > A mutation is homozygous in patients (Patient), heterozygous in parents and some of the unaffected siblings (Carriers) and absent in 100 normal controls (Normal)

Article Snippet: The blots were blocked in 5 % milk in Phosphate Buffered Saline with Tween 20 (PBST) and probed with rabbit anti-ELAC2 antibody (1:100; sc-138774, Santa Cruz, USA) overnight.

Techniques: Genome Wide, Sequencing, Mutagenesis

Journal: Orphanet Journal of Rare Diseases

Article Title: A homozygous splicing mutation in ELAC2 suggests phenotypic variability including intellectual disability with minimal cardiac involvement

doi: 10.1186/s13023-016-0526-8

Figure Lengend Snippet: Effect of the splicing mutation on ELAC2 expression and on selected mitochondrial genes. a ) The amplification products of ELAC2 cDNA from patients, controls and parents were seen on a 2 % agarose gel. Bright bands were detected in the lanes of two healthy controls (C1 and C2) and the mother (IV4) at 500 bp (according to the DNA size marker M). Multiple fainter bands were seen in the patients’ (V10 and V11) lanes suggesting diminished expression of the normal WT transcript and the presence of other abnormal splicing products. b ) Expression analysis of ELAC2 protein in patient fibroblasts. Total protein lysates from patient (V10) and two different control fibroblasts were analyzed for ELAC2 protein expression by immunoblotting against an antibody specific for ELAC2 isoform1. HEK293T cell lysate was used as a positive control. Mouse Tubulin antibody was used as a loading control. The levels of the protein was negligible in patient fibroblast compared to the control. Densitometric analysis of ELAC2 protein bands normalized to Tubulin levels, revealed that ELAC2 protein expression in patient fibroblasts was 14 % of that detected in control fibroblasts. c ) A significant difference is seen between relative expressions of different unprocessed mitochondrial transcripts mtATP8, mtND2, and mtND4 , in skin fibroblasts from patient V10, compared to four different control samples (Ctl1, Ctl2, Ctl5, and Ctl6). The mRNA expression values were normalized to an internal control HPRT. X axis depicts quantitative expression; Y axis represents bar chart for controls, and patient samples, respectively

Article Snippet: The blots were blocked in 5 % milk in Phosphate Buffered Saline with Tween 20 (PBST) and probed with rabbit anti-ELAC2 antibody (1:100; sc-138774, Santa Cruz, USA) overnight.

Techniques: Mutagenesis, Expressing, Amplification, Agarose Gel Electrophoresis, Marker, Control, Western Blot, Positive Control

Journal: Orphanet Journal of Rare Diseases

Article Title: A homozygous splicing mutation in ELAC2 suggests phenotypic variability including intellectual disability with minimal cardiac involvement

doi: 10.1186/s13023-016-0526-8

Figure Lengend Snippet: Summary of clinical features of patients with ELAC2 mutations

Article Snippet: The blots were blocked in 5 % milk in Phosphate Buffered Saline with Tween 20 (PBST) and probed with rabbit anti-ELAC2 antibody (1:100; sc-138774, Santa Cruz, USA) overnight.

Techniques:

Journal: Nucleic acids research

Article Title: A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism.

doi: 10.1093/nar/gkac720

Figure Lengend Snippet: Figure 6. In vitro assay for the processing of mitochondrial tRNACys precursors. (A, B) tRNACys precursors for 5′ or 3′ end processing assays. Thirty-six nucleotides (nt) of 5′ and 3′ end leaders of tRNACys were shown, including the m.5783C > T substitution. (C) In vitro 5′ end processing assays. Processing assays with mitochondrial RNase P were carried out in parallel for wild type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent sub- strate CDP-Star™to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. (D) Relative processing efficiencies of tRNACys precursors catalyzed by RNase P. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. (E) In vitro 3′ end processing assays. Processing assays with mitochondrial RNase Z were undertaken in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. (F) Quantification of the efficiencies of tRNACys precursors catalyzed by RNase Z. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. (G) Western blot analysis of tRNA processing related proteins MRPP2, ELAC2 and TRNT1 with β-Actin as a loading control. (H) Quantification of MRPP2, ELAC2 and TRNT1. Average relative values of MRPP2, ELAC2 and TRNT1 were normalized to the average values of β-Actin in various cell lines. The values for the latter are expressed as percentages of the average values for the control cell lines. The average of three independent determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 2.

Article Snippet: The antibodies used for this investigation were from Abcam [TOM20 (ab56783), Total OXPHOS Human WB Antibody Cocktail (ab110411), Proteintech [β-actin (20536-1-AP), Twinkle (13435-1-AP), SSBP1 (12212-1-AP), ABclonal [Pol A (A1323), ELAC2 (A7128), TRNT1 (A17699), D ow nloaded from https://academ ic.oup.com /nar/article/50/16/9453/6678873 by guest on 05 February 2024 MRPP2 (A0959)], Invitrogen [POLRMT (PA5-28196)] and BOSTER [TFAM (BA2827)].

Techniques: In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Western Blot, Control

Journal: Frontiers in Molecular Biosciences

Article Title: ELAC2, an Enzyme for tRNA Maturation, Plays a Role in the Cleavage of a Mature tRNA to Produce a tRNA-Derived RNA Fragment During Respiratory Syncytial Virus Infection

doi: 10.3389/fmolb.2020.609732

Figure Lengend Snippet: ELAC2 is responsible for the induction of tRF5-GlnCTG. (A) A549 cells were transfected with 100 nmol/l of siRNA against individual indicated nuclease or control siRNA. At 40 h post-transfection, the cells were mock- or RSV-infected at an MOI of 1 for 24 h, followed by Cyto RNA preparation and Northern hybridization for the detection of tRF5-GlnCTG. The individual gene knockdown was verified by qRT-PCR (down panel). (B) Cells were transfected with siRNAs and then mock- or RSV-infected as described in (A) , followed by total protein preparation and then Western blot using antibodies against individual targets. β-actin was used as a loading control. (C) Compartmental locations of ELAC2. Cells were uninfected or infected with RSV at an MOI of 1 for 24 h, followed by subcellular fractionation preparation for mitochondria-free cytoplasm (Mito-free Cyto), nuclei (Nu), and mitochondria (Mito) and then Western blot using the indicated antibodies. The purity of fractions was assessed by compartment-specific proteins. The succinate dehydrogenase complex subunit A (SDHA), Lamin B1, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serve as a mitochondrial, nuclear, and mitochondria-free cytoplasmic marker, respectively. Data are representative of 2-3 independent experiments. (D) RNA was prepared from subcellular fractionated compartments as described in (C) . The same amount of 10 µg in all fractionated RNAs was loaded to a denaturing polyacrylamide gel for Northern hybridization to detect tRF5-GlnCTG. The loading ratio of each fraction was: all Mito RNAs vs 1/25 for Nu RNA vs 1/100 for Mito-free Cyto RNA. Data are representative of 2-3 independent experiments.

Article Snippet: The antibodies were purchased from the following manufacturers: ANG (sc-74528; Santa Cruz Biotechnology, Santa Cruz, CA), Drosha (sc-393591; Santa Cruz Biotechnology), and Lamin B1 (sc-374015; Santa Cruz Biotechnology), Horseradish-coupled secondary antibodies (sc-2031 and sc-2030; Santa Cruz Biotechnology), β-actin (A1978; Sigma-Aldrich), GAPDH (G9545; Biotrend chemicals, Köln, Germany), Dicer (5362T; Cell Signaling Technology, Danvers, MA), SDHA (5839; Neobiolab, Woburn, MA), RNase L (MBS8292747; MyBioSource, San Diego, CA), ELAC2 (A304-775A-T; Bethyl Laboratories, Montgomery, TX), and V5 (MCA1360GA; Bio-rad).

Techniques: Transfection, Control, Infection, Northern Blot, Hybridization, Knockdown, Quantitative RT-PCR, Western Blot, Fractionation, Marker

Journal: Frontiers in Molecular Biosciences

Article Title: ELAC2, an Enzyme for tRNA Maturation, Plays a Role in the Cleavage of a Mature tRNA to Produce a tRNA-Derived RNA Fragment During Respiratory Syncytial Virus Infection

doi: 10.3389/fmolb.2020.609732

Figure Lengend Snippet: The contribution of RSV proteins to the induction of tRF5-GlnCTG. (A) A549 cells in triplicate were transfected with plasmids encoding V5-tagged RSV N, P, NS1, NS2, or empty vector (CN, negative control for viral protein expression). Vectors of Pp -anti_GlnCTG_WT (containing the target sequence of tRF5-GlnCTG) or Pp -anti_Control (without tRF5-GlnCTG target sequence) were also co-transfected with renilla luciferase plasmids ( Rr , luciferase expression internal control). At 30 h post-transfection, the cells were harvested for the dual-luciferase assay. Pp luciferase values were normalized to their corresponding Rr luciferase values and then the normalized value of Pp -anti_GlnCTG_WT was normalized to that of Pp -anti_Control vector. Data are representative of three independent experiments. ** P < 0.01, relative to the first bar. (B) 293 cells were transfected with individual V5-tagged viral proteins as indicated. CN and P-expressing plasmids were used as controls. After 30 h, the cells were harvested and immunoprecipitation was done using the anti-V5 antibody, followed by Western blot using the anti-ELAC2 and anti-V5 antibodies. Proper expression of ELAC2 and V5-tagged RSV proteins was confirmed in sample input. Data are representative of three independent experiments.

Article Snippet: The antibodies were purchased from the following manufacturers: ANG (sc-74528; Santa Cruz Biotechnology, Santa Cruz, CA), Drosha (sc-393591; Santa Cruz Biotechnology), and Lamin B1 (sc-374015; Santa Cruz Biotechnology), Horseradish-coupled secondary antibodies (sc-2031 and sc-2030; Santa Cruz Biotechnology), β-actin (A1978; Sigma-Aldrich), GAPDH (G9545; Biotrend chemicals, Köln, Germany), Dicer (5362T; Cell Signaling Technology, Danvers, MA), SDHA (5839; Neobiolab, Woburn, MA), RNase L (MBS8292747; MyBioSource, San Diego, CA), ELAC2 (A304-775A-T; Bethyl Laboratories, Montgomery, TX), and V5 (MCA1360GA; Bio-rad).

Techniques: Transfection, Plasmid Preparation, Negative Control, Expressing, Sequencing, Control, Luciferase, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism

doi: 10.1093/nar/gkac720

Figure Lengend Snippet: In vitro assay for the processing of mitochondrial tRNA Cys precursors. ( A, B ) tRNA Cys precursors for 5′ or 3′ end processing assays. Thirty-six nucleotides (nt) of 5′ and 3′ end leaders of tRNA Cys were shown, including the m.5783C > T substitution. ( C ) In vitro 5′ end processing assays. Processing assays with mitochondrial RNase P were carried out in parallel for wild type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™ to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. ( D ) Relative processing efficiencies of tRNA Cys precursors catalyzed by RNase P. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. ( E ) In vitro 3′ end processing assays. Processing assays with mitochondrial RNase Z were undertaken in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™ to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. ( F ) Quantification of the efficiencies of tRNA Cys precursors catalyzed by RNase Z. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. ( G ) Western blot analysis of tRNA processing related proteins MRPP2, ELAC2 and TRNT1 with β -Actin as a loading control. ( H ) Quantification of MRPP2, ELAC2 and TRNT1. Average relative values of MRPP2, ELAC2 and TRNT1 were normalized to the average values of β -Actin in various cell lines. The values for the latter are expressed as percentages of the average values for the control cell lines. The average of three independent determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Abcam [TOM20 (ab56783), Total OXPHOS Human WB Antibody Cocktail (ab110411), Proteintech [ β -actin (20536-1-AP), Twinkle (13435-1-AP), SSBP1 (12212-1-AP), ABclonal [PolγA (A1323), ELAC2 (A7128), TRNT1 (A17699), MRPP2 (A0959)], Invitrogen [POLRMT (PA5-28196)] and BOSTER [TFAM (BA2827)].

Techniques: In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism

doi: 10.1093/nar/gkac720

Figure Lengend Snippet: In vitro assay for the processing of mitochondrial tRNA Cys precursors. ( A, B ) tRNA Cys precursors for 5′ or 3′ end processing assays. Thirty-six nucleotides (nt) of 5′ and 3′ end leaders of tRNA Cys were shown, including the m.5783C > T substitution. ( C ) In vitro 5′ end processing assays. Processing assays with mitochondrial RNase P were carried out in parallel for wild type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™ to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. ( D ) Relative processing efficiencies of tRNA Cys precursors catalyzed by RNase P. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. ( E ) In vitro 3′ end processing assays. Processing assays with mitochondrial RNase Z were undertaken in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™ to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. ( F ) Quantification of the efficiencies of tRNA Cys precursors catalyzed by RNase Z. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. ( G ) Western blot analysis of tRNA processing related proteins MRPP2, ELAC2 and TRNT1 with β -Actin as a loading control. ( H ) Quantification of MRPP2, ELAC2 and TRNT1. Average relative values of MRPP2, ELAC2 and TRNT1 were normalized to the average values of β -Actin in various cell lines. The values for the latter are expressed as percentages of the average values for the control cell lines. The average of three independent determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Abcam [TOM20 (ab56783), Total OXPHOS Human WB Antibody Cocktail (ab110411), Proteintech [ β -actin (20536-1-AP), Twinkle (13435-1-AP), SSBP1 (12212-1-AP), ABclonal [PolγA (A1323), ELAC2 (A7128), TRNT1 (A17699), MRPP2 (A0959)], Invitrogen [POLRMT (PA5-28196)] and BOSTER [TFAM (BA2827)].

Techniques: In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Western Blot, Control