Additional file 2: of DensityMap: a genome viewer for illustrating the densities of features

DensityMap Manual. (DOCX 239 kb

[Hosta sp.]

原著和名: [記載なし]科名: ユリ科 = Liliaceae採集地: 宮崎県 東臼杵郡 大崩山 (日向 東臼杵郡 大崩山)採集日: 1973/8/1採集者: 萩庭丈壽整理番号: JH003752国立科学博物館整理番号: TNS-VS-953752備考: DB作成協力会による補足あ

Additional file 8: of Deep landscape update of dispersed and tandem repeats in the genome model of the red jungle fowl, Gallus gallus, using a series of de novo investigating tools

Diversity of CR1 within galGal4. The clustering of CR1 copies into subfamilies was re-investigated using SiLiX. Eight subfamilies were found, 7 of them matching with the Repbase sub-families CR1-C, CR1-D, CR1_F, CR1-G, CR1_GG, CR1-H, and CR1-Y. Their respective abundance in galGal4 was summarized in Table S2. (ODT 30 kb

Expression (A) and kinetic (B) of <i>PB</i> transposase by type and quantity of nucleic acid.

<p>HeLa cells were transfected with indicated amounts (A) or 187.5 ng (B) of <i>PB</i> mRNA or pDNA and total protein extraction was performed 24 h (A) or at indicated times (B) post-transfection. Transposase (V5PB Tp) expression was determined by Western-Blot and protein quantification was normalized to the endogenous Menin protein. Values represent the average of 3 experiments done in triplicate. Mock: untransfected cells. * Indicates statistically significant differences between mRNA and pDNA (p<0.05).</p

Transposition assay in mammalian cells.

<p>(A) Donor plasmid harboring the transposable element. This vector is composed of the neomycin phosphotransferase gene (Neo^R) flanked by the <i>PB</i> ITRs. (B) Transposition efficiency by type and quantity of nucleic acid. Cells were transfected with indicated amounts of <i>PB</i> mRNA or pDNA alongside with 187.5 ng of donor plasmid. GFP mRNA or pDNA (500 ng) served as negative controls of transposition (Mock: recombination events). To consider only transposition events, the number of colonies observed in the presence of the transposase source was subtracted by the number of colonies obtained in the respective negative control performed without transposase for each quantity. Values represent the average of 3 experiments done in triplicate. * Represents statistically significant difference between mRNA and pDNA (p<0.05).</p

Detection of H2AX phosphorylation following dose-dependent <i>PB</i> mRNA or pDNA transfection.

<p>Cells were transfected with indicated amounts of <i>PB</i> mRNA or pDNA and total protein extraction was performed 24 h post-transfection. γ-H2AX expression was determined by Western-Blot and protein quantification was normalized to the endogenous Menin protein. Mock: untransfected cells. T-_GFP: cells transfected with 500 ng of GFP mRNA or pDNA. T+: untransfected cells treated with 2 µg/mL doxorubicin (positive control). * Indicates statistically significant differences between treated and untreated cells (p<0.05). Values represent the average of 3 experiments done in triplicate. The signal between the dotted line (mock control) and solid line (T-_GFP control) is considered to be due to the “transfection effect”. Above the solid line, the signal is due to the “transposase effect”.</p

<i>piggyBac</i> transposase bioavailability.

<p>(<b>A</b>) <b>Half-life of the <i>PB</i> mRNA</b>. Cells were transfected with 187.5 ng of <i>PB</i> mRNA. Total RNA was extracted at indicated times, reverse transcribed and subjected to qPCR. 18S RNA served as an internal standard to normalize the data. (<b>B</b>) <b>Persistence of <i>PB</i> pDNA after transfection</b>. Cells were transfected with 187.5 ng of <i>PB</i> pDNA and plasmid rescue was performed at 0 to 20 days. Ampicillin-resistant colonies were selected to evaluate the persistence of the plasmid. (<b>C</b>) <b>Half-life of the <i>PB</i> transposase (V5PB Tp)</b>. Cells were transfected with 187.5 ng of <i>PB</i> mRNA, incubated 18 h to reach the peak of transposase expression and treated with cycloheximide (t0=100). Total protein extraction was done at the indicated times from t0. The transposase half-life was determined by Western-Blot and quantification was normalized to the endogenous actin protein.</p

Perturbation of mRNP biogenesis reveals a dynamic landscape of the Rrp6-dependent surveillance machinery trafficking along the yeast genome

<p>Eukaryotic cells have evolved a nuclear quality control (QC) system to monitor the co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent ribonucleoprotein particles (mRNPs). Aberrant mRNPs that fail to pass the QC steps are retained in the nucleus and eliminated by the exonuclease activity of Rrp6. It is still unclear how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events that may arise at each step of transcript elongation and mRNP formation. To dissect the QC mechanism, we previously implemented a powerful assay based on global perturbation of mRNP biogenesis in yeast by the bacterial Rho helicase. By monitoring model genes, we have shown that the QC process is coordinated by Nrd1, a component of the NNS complex (Nrd1-Nab3-Sen1) involved in termination, processing and decay of ncRNAs which is recruited by the CTD of RNAP II. Here, we have extended our investigations by analyzing the QC behaviour over the whole yeast genome. We performed high-throughput RNA sequencing (RNA-seq) to survey a large collection of mRNPs whose biogenesis is affected by Rho action and which can be rescued upon Rrp6 depletion. This genome-wide perspective was extended by generating high-resolution binding landscapes (ChIP-seq) of QC components along the yeast chromosomes before and after perturbation of mRNP biogenesis. Our results show that perturbation of mRNP biogenesis redistributes the QC components over the genome with a significant hijacking of Nrd1 and Nab3 from genomic loci producing ncRNAs to Rho-affected protein-coding genes, triggering termination and processing defects of ncRNAs.</p

Location of regions essential to silencing activity within the Δ8 segments of <i>MOS1</i>.

<p>(<b>a</b>) Variant fragments within the sequences of the Δ8-<i>MOS1</i>. Dashes indicate positions that are present in each fragment. (<b>b</b>) Expression of the <i>Firefly</i> and the <i>Renilla</i> luciferase marker genes using transient expression assays in HeLa cells. The assays were performed with Δ8-<i>MOS1</i> and five variants cloned in + orientation. (<b>c</b>) and (<b>d</b>) Impact of the Δ7 segments of <i>MOS1</i> on the expression of the <i>Firefly</i> and the <i>Renilla</i> luciferase marker genes using transient expression assays in cell lineages H4 (TARBP2+/NRSF-/YY1+/NFAT-5-) (<b>c</b>) and DT40 (TARBP2+/NRSF+/YY1+/NFAT-5-) (<b>d</b>). In (<b>b</b>), (<b>c</b>) and (<b>d</b>), each histogram bar corresponds to the median value obtained from three experiments done in triplicate. Bars correspond to quartiles 1 and 3. The median ratio RLU from <i>Firefly</i>/RLU from <i>Renilla</i> were calculated as indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005902#pgen.1005902.g002" target="_blank">Fig 2</a>. The area where the ratios “RLU from <i>Firefly</i>/RLU from <i>Renilla</i>” were above 1 (i.e. where no strong silencer effect is observed) is coloured in grey. In (<b>b</b>) * and ** indicate a significant difference (p<0.05) with the P_Luc controls. ** also indicates a significant difference (p<0.05)with HS2_P_Luc_Δ8-<i>MOS1</i>.</p

Characterization of a <i>Mos1</i> region that negatively interferes with the expression of a marker gene.

<p>(<b>a</b>) <i>Mos1</i> transposon and the various deletion derivatives used herein (Δ1 to Δ7). Blank rectangle: <i>Mos1</i> transposase Open Reading Frame (Tpase ORF). Black arrow: 5’ ITR (Inverted Terminal Repeat) fused to the 5’ UTR (UnTranslated Region). Grey arrow: 3’ UTR fused to the 3’ ITR. Δ1 spanned from positions 1 to 655, Δ2 from 656 to 1289, Δ3 from 1 to 174, Δ4 from 1213 to 1289, Δ5 from 1 to 1212, Δ6 from 175 to 1289, and Δ7 from 681 to 1212 of the <i>Mos1</i> sequence (Acc N°X78906; 1289 bp). (<b>b</b>), (<b>c</b>), (<b>d</b>), and (<b>e</b>) Stable expression assays using the neomycin resistance gene marker in HeLa cells. Different constructs composed of the <i>NeoR</i> marker flanked with <i>Mos1</i> segments presented in (<b>a</b>) at one or both ends were transfected in HeLa cells. Stable integrants were obtained following 15 days of antibiotic selection and resistant colonies were counted. (<b>b</b>) Characterization of the segment in the <i>Mos1</i> transposon able to silence the expression of the neomycin resistance gene. (<b>c</b>) and (<b>d</b>) Stained stable integrants obtained after transfection with the control [<i>NeoR</i>] construct (~ 275–300 clones) and the [<i>NeoR</i>]Δ7 construct (~ 25–30 clones). (<b>e</b>) Effects of the location and orientation of the Δ7 segment on the marker gene expression. The Δ7 segment was placed upstream or downstream of the marker gene in the positive (+) or negative (-) orientation. (<b>f</b>) Impact of the Δ7-<i>MOS1</i> segment intragenic location using a transient gene expression assay. Δ7 and Δ1b were fused in frame to the 5’ end of the <i>GFP</i> gene in pCS2+ expression plasmids (pCMV promoter) as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005902#pgen.1005902.ref012" target="_blank">12</a>]. Segment Δ1b (= Δ1 without the 5’ITR-UTR region) or the full length <i>MOS1</i> ORF were used as controls. Each construct was transfected into HeLa cells and GFP expression was analysed by fluorescent flow cytometry and compared to HeLa cells transfected with a pCS2-GFP plasmid. Median values from three experiments performed in triplicate are shown. Bars correspond to quartiles 1 and 3. The median value obtain with the control [<i>NeoR</i>] construct was fixed at 100% and used as a reference to calculate the medians of the other constructs. Results obtained with [<i>NeoR</i>] (white bar) and Δ1[<i>NeoR</i>] Δ2 (black bar) were used as benchmarks for all our stable expression assays. * indicates significant difference (p<0.05) compared to the [<i>NeoR</i>] or <i>GFP</i> control.</p