RNase protection assay on mitochondrial (−)strand transcripts mapping around the cyt b/ND1-binding site in DmTTF-depleted cells

<p><b>Copyright information:</b></p><p>Taken from "The termination factor DmTTF regulates mitochondrial transcription"</p><p>Nucleic Acids Research 2006;34(7):2109-2116.</p><p>Published online 28 Apr 2006</p><p>PMCID:PMC1450328.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Schematic representation of digestion products using probes Ribo-1 (295 nt) and Ribo-2 (218 nt). Riboprobes (grey bold arrows), mature transcripts (continuous arrows) and read-through transcripts (dotted arrows) are indicated above the cyt b/ND1 region map. Dashed regions indicate non-coding sequences. () Total cellular RNA (50 µg), extracted from untreated (control) and DmTTF-dsRNA treated (RNAi) D.Mel-2 cells, was hybridized with about 1.5 × 10 c.p.m. of Ribo-1 and Ribo-2 probes and digested with RNase A and T1. Digestion products were denatured and run on a 10% polyacrylamide/7 M urea gel. Y, sample containing 50 µg of yeast total RNA. M, Decade RNA marker (Ambion)

RT–PCR analysis on mitochondrial transcripts spanning the cyt b/ND1-binding site in DmTTF-depleted cells

<p><b>Copyright information:</b></p><p>Taken from "The termination factor DmTTF regulates mitochondrial transcription"</p><p>Nucleic Acids Research 2006;34(7):2109-2116.</p><p>Published online 28 Apr 2006</p><p>PMCID:PMC1450328.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Schematic illustration of the cyt b/ND1-binding site. Black arrows represent the forward (11 494–11 520 nt) and reverse (11 855–11 834 nt) primers used for RT–PCR. Dashed regions indicate non-coding nucleotides. () Total RNA (600 ng) extracted from untreated (control) and treated (RNAi) D.Mel-2 cells was used as template in RT–PCR; 10 µl-samples were collected at the indicated cycles, run on a 1.5% agarose gel and stained with ethidium bromide. Nuclear encoded 28S rRNA was used as endogenous control

Purification of the sea urchin mtRNAP from baculovirus-infected insect cells and functional assay

<p><b>Copyright information:</b></p><p>Taken from "Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system"</p><p></p><p>Nucleic Acids Research 2007;35(7):2413-2427.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874651.</p><p>© 2007 The Author(s)</p> () Purification of mtRNAP by metal chelate affinity chromatography. The soluble portion of the insect cell lysate expressing the sea urchin mtRNAP was purified by Ni-NTA chromatography; cleared lysate, C.lys, flow-through, FT, wash, W, 3–5, fractions eluted at 250 mM imidazole, were separated on a 10% SDS–PAGE and revealed by immunoblotting as described in ‘Materials and Methods’ section. () Purification profile of mtRNAP as obtained by Heparin–Sepharose chromatography. Peak fractions from Ni-NTA column were pooled and subjected to Heparin–Sepharose chromatography. Input to the column (I) and fractions eluting between 0.75 and 0.9 M NaCl were analyzed by 7.5% SDS–PAGE and Coomassie Brilliant Blue stained. The molecular weight marker Precision Plus Protein Standards (Bio-Rad) is shown (M). The arrow inside the picture indicates the mtRNAP-containing band, as assessed by MALDI-TOF analysis. () Immunoblotting assay of input to the column (I) and Heparin–Sepharose eluted fractions. () Transcriptional activity of purified mtRNAP. The indicated Heparin–Sepharose fractions were assayed in the presence of [α-]PUTP, as described in ‘Materials and Methods’ section. On the top it is shown the diagram of the 71-bp tailed template, named 71bpDNA, with the open bar referring to the duplex DNA portion and the thin line to the 3′-tail. Run-off transcripts are indicated by arrowed line. Radiolabeled transcripts were separated on a 12% polyacrylamide/7M urea mini-gel followed by phosphorimaging analysis. 15 + R, fraction 15 treated with RNase A. RNA markers corresponding to the 10 nt ladder are indicated on the left

Conditional inactivation of the <i>Nsun4</i> gene in the germline.

<p>A. Schematic representation of the targeting strategy for disruption of the <i>Nsun4</i> gene. Exon II was flanked by loxP-sites. The puromycin resistance gene is flanked by Frt-recombination sites and was used for ES cell selection. Mice with an Frt-flanked puromycin gene were crossed with mice with ubiquitous expression of Flp-recombinase to remove the Puromycin resistance gene. Transgenic mice expressing cre-recombinase were used for breeding with animals with a loxP-flanked <i>Nsun4</i> gene to disrupt <i>Nsun4</i>. B. Morphological comparison between wild type and whole-body <i>Nsun4</i> knockout embryos at E∼8.5. Scale bar = 1 mm.</p

rRNA binding by NSUN4 and MTERF4 and model of the role of the NSUN4/MTERF4 complex in regulation of ribosome assembly.

<p>A. Location, along mtDNA, of the RNA fragments identified after CLIP analysis on HeLa cells expressing “trap” mutant of NSUN4, NSUN4^C258A-FLAG. B. Location, along mtDNA, of the RNA fragments identified after PAR-CLIP analysis on HeLa cells expressing MTERF4-FLAG. C. Model for the role of NSUN4 in mitoribosomal assembly. (1) NSUN4 methylates 12S rRNA. Red star denotes the methyl group donated by SAM. (2) The NSUN4/MTERF4 complex is incorporated into the LSU. (3) Release of the NSUN4/MTERF4 complex from LSU enables the interaction between both subunits.</p

Conditional inactivation of the <i>Nsun4</i> gene in heart and skeletal muscle.

<p>A. Decreased lifespan of mice with a muscle-specific knockout of <i>Nsun4</i>. Survival curve for control (L/L; n = 20; open squares) and mutant mice (L/L, cre; n = 16; filled squares). B. Heart- to body-weight ratio in control (L/L; open squares) and mutant mice (L/L, cre; filled squares). Number of analyzed animals at 5 weeks L/L n = 6, L/L, cre n = 6; at 10 weeks L/L n = 7, L/L, cre n = 8; at 15 weeks L/L n = 5, L/L, cre n = 7; at 20 weeks L/L n = 12, L/L, cre n = 12. Data are represented as mean +/− SEM. *, p<0.05; **, p<0.01; ***p<0.001. Student's t test. C. BN-PAGE analysis of levels of assembled respiratory chain complexes in control (L/L) and knockout (L/L, cre) mice at different ages. D. Western immunoblotting of steady-state levels of NSUN4 in heart mitochondrial extracts from control (L/L) and knockout (L/L, cre) mice at different ages. VDAC was used as a loading control.</p

Mitochondrial translation and ribosome assembly in <i>Nsun4</i> knockout hearts.

<p>A. Pulse-labeling of mitochondrial translation products in isolated heart mitochondria from 20 weeks-old control (L/L) and knockout mice (L/L, cre). The Coomassie-stained gel is a loading control. Known mitochondrial polypeptides are indicated. B. Western blot analysis of steady-state levels of mitochondrially and nucleus-encoded OXPHOS proteins in mitochondria from control and knockout hearts at different ages. VDAC was used as a loading control. *, cross reaction. C. Analysis of mitoribosomal assembly by sucrose gradient ultracentrifugation of heart mitochondrial extracts from control (L/L) and mutant (L/L, cre) mice. Sedimentation of 28S (SSU, fraction 6), 39S (LSU, fraction 8) and 55S (assembled ribosomes, fraction 10) was determined by western blot analysis using MRPS15- and MRPL13-specific antibodies. D. Western blot analysis of steady-state levels of MRPL13 and MRPS15 in heart mitochondrial extracts from control (L/L) and knockout (L/L, cre) mice at different ages. VDAC was used as a loading control.</p

Analysis of rRNA methylation in control, <i>Nsun4</i> and <i>Mterf4</i> heart tissue specific knockout.

<p>A. Relative methylation levels of 12S and 16S rRNA determined after sequencing of cDNA obtained from bisulfite treated RNA from heart mitochondria of control (L/L; n = 3) mice at age 20 weeks. Nucleotide numbers are relative to the 5′-end of the mouse mtDNA gene for tRNA^Phe. B. Relative methylation levels of 12S and 16S rRNA in NSUN4 knockout (L/L, cre; n = 3) at age 20 weeks. Analysis performed as in panel a. C. Relative methylation levels of 12S rRNA in control (N = 1) and MTERF4 knockout (N = 2) at age 15 weeks and in control (N = 1) and MTERF4 knockout (N = 2) at age 20 weeks.</p

Creation and characterization of <i>DmMterf3</i> knockout larvae.

<p>(A) The <i>DmMterf3</i> locus and generation of a <i>DmMterf3</i> null mutant. <i>DmMterf3</i> is located on the third chromosome at cytological position 77C6. The construct (ko <i>DmMterf3</i>) used for ends-out homologous recombination is indicated by grey boxes, coding sequences in exons are indicated by black boxes and non-coding sequences in exons by white boxes. The gap between grey boxes represents the genomic region replaced by attP and a <i>white</i> marker gene. The white marker gene was subsequently removed by crossing to <i>cre</i>-recombinase expressing flies. (B) PCR analysis of wild-type and knockout alleles for <i>DmMterf3</i>. (C) Body size comparison of <i>DmMterf3</i> knockout larvae 6 days after egg-laying (ael) showing reduced size. (D) QRT-PCR analysis of <i>DmMterf3</i> transcript levels in control (white and grey bars) and <i>DmMterf3</i> KO larvae (black bars) at 3 and 6 days ael. (E) Q-PCR analysis of mtDNA levels in larvae at 3 and 6 days ael. (F) QRT-PCR analysis of steady-state levels of mitochondrial mRNAs normalized to the nuclear ribosomal protein 49 transcript levels in larvae at 3 and 6 days ael. Error bars indicate mean ± SEM (^*p<0.05; ^**p<0.01; ^***p<0.001; n = 5).</p

Binding of MTERF3 to mitochondrial 16S rRNA and model of MTERF3 action.

<p>(A) Identification of mitochondrial transcripts interacting with MTERF3 in mice was performed by co-immuno-precipitation using a mouse anti-MTERF3 antibody. Transcripts bound to mouse MTERF3 were quantified using qRT-PCR and their abundance is shown as percentage of levels in control IPs. (B) Identification of mitochondrial transcripts interacting with MTERF3 in flies was performed by co-immuno-precipitation using an anti-FLAG antibody and extracts from flies expressing DmMTERF3-linker-Flag. Transcripts bound to fly MTERF3 were quantified using qRT-PCR and their abundance is shown as percentage of levels in control IPs. Error bars correspond to standard deviation. (C) Model of the physiological role of MTERF3 in control of mtDNA expression (left panel) and model of the phenotypes caused by loss of MTERF3 (right panel).</p