Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals
Publication Date
February 13, 2015
Journal
PLOS Genetics
Authors
Jules Gagnon, Mathieu Lavoie, Mathieu Catala, Francis Malenfant, et al
Volume
11
Issue
2
Pages
e1005000
DOI
https://dx.plos.org/10.1371/journal.pgen.1005000
Publisher URL
http://journals.plos.org/plosgenetics/article?id=10.1371%2Fjournal.pgen.1005000
Web of Science
000352081800053
Scopus
84924354128
Mendeley
http://www.mendeley.com/research/transcriptome-wide-annotation-eukaryotic-rnase-iii-reactivity-degradation-signals
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Mendeley | Further Information

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Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1930689"], "description"=>"<p>(<b>A</b>) Deletion of <i>RNT1</i> induces global perturbation of the yeast transcriptome. The levels of gene expression in wild type (WT) cells, <i>rnt1∆</i> (top) and <i>rrp6∆</i> (bottom) strains were determined using tiling arrays covering the entire yeast genome and presented in the form of dot plot comparison. Dots corresponding to the expression values of protein-coding genes, non-coding RNA and intergenic regions are shown in black, red and green, respectively <b>(<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s014\" target=\"_blank\">S7 Table</a>)</b>. Blue crosses highlight the expression of the known Rnt1p substrates as indicated in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.g001\" target=\"_blank\">Fig. 1A</a></b>. (<b>B</b>) Rnt1p regulates the expression of both coding and non-coding genes. Pie chart illustrating the types of RNAs upregulated by at least two folds upon <i>RNT1</i> deletion (details in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s016\" target=\"_blank\">S9 Table</a></b>). Multiple transcripts within the same overexpressed segments were counted individually. PCGs, ncRNAs and LTRs indicate protein-coding genes, non-coding RNAs and long terminal repeats, respectively. (<b>C</b>) Expression of non-coding RNAs (ncRNAs) is highly sensitive to <i>RNT1</i> deletion. The top 30 upregulated genes in <i>rnt1∆</i> cells were sorted according to their RNA types (details in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s017\" target=\"_blank\">S10 Table</a></b>). (<b>D</b>) RNAs upregulated by the deletion of <i>RNT1</i> resist cleavage <i>in vitro</i>. Northern blot analysis of RNA extracted from wild type (<i>RNT1</i>) and <i>rnt1∆</i> cells before and after incubation with Rnt1p (<i>rnt1∆</i>+Rnt1p) was performed as described in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.g001\" target=\"_blank\">Fig. 1G</a></b>. Cleavage was visualized using probes complementary to the sequence of HSP12 (left), YGP1 (middle) or RTC3 (right) mRNAs.</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322624, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g002", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gene_expression_is_a_poor_indicator_of_substrate_reactivity_/1322624", "title"=>"Gene expression is a poor indicator of substrate reactivity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930724", "https://ndownloader.figshare.com/files/1930725", "https://ndownloader.figshare.com/files/1930726", "https://ndownloader.figshare.com/files/1930727", "https://ndownloader.figshare.com/files/1930728", "https://ndownloader.figshare.com/files/1930729", "https://ndownloader.figshare.com/files/1930730", "https://ndownloader.figshare.com/files/1930731", "https://ndownloader.figshare.com/files/1930732", "https://ndownloader.figshare.com/files/1930733", "https://ndownloader.figshare.com/files/1930734", "https://ndownloader.figshare.com/files/1930735", "https://ndownloader.figshare.com/files/1930736", "https://ndownloader.figshare.com/files/1930737", "https://ndownloader.figshare.com/files/1930738", "https://ndownloader.figshare.com/files/1930739", "https://ndownloader.figshare.com/files/1930740", "https://ndownloader.figshare.com/files/1930741", "https://ndownloader.figshare.com/files/1930742", "https://ndownloader.figshare.com/files/1930743", "https://ndownloader.figshare.com/files/1930744", "https://ndownloader.figshare.com/files/1930745", "https://ndownloader.figshare.com/files/1930746", "https://ndownloader.figshare.com/files/1930747", "https://ndownloader.figshare.com/files/1930748", "https://ndownloader.figshare.com/files/1930749", "https://ndownloader.figshare.com/files/1930750"], "description"=>"<div><p>Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions.</p></div>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322651, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>["https://dx.doi.org/10.1371/journal.pgen.1005000.s001", "https://dx.doi.org/10.1371/journal.pgen.1005000.s002", "https://dx.doi.org/10.1371/journal.pgen.1005000.s003", "https://dx.doi.org/10.1371/journal.pgen.1005000.s004", "https://dx.doi.org/10.1371/journal.pgen.1005000.s005", "https://dx.doi.org/10.1371/journal.pgen.1005000.s006", "https://dx.doi.org/10.1371/journal.pgen.1005000.s007", "https://dx.doi.org/10.1371/journal.pgen.1005000.s008", "https://dx.doi.org/10.1371/journal.pgen.1005000.s009", "https://dx.doi.org/10.1371/journal.pgen.1005000.s010", "https://dx.doi.org/10.1371/journal.pgen.1005000.s011", "https://dx.doi.org/10.1371/journal.pgen.1005000.s012", "https://dx.doi.org/10.1371/journal.pgen.1005000.s013", "https://dx.doi.org/10.1371/journal.pgen.1005000.s014", "https://dx.doi.org/10.1371/journal.pgen.1005000.s015", "https://dx.doi.org/10.1371/journal.pgen.1005000.s016", "https://dx.doi.org/10.1371/journal.pgen.1005000.s017", "https://dx.doi.org/10.1371/journal.pgen.1005000.s018", "https://dx.doi.org/10.1371/journal.pgen.1005000.s019", "https://dx.doi.org/10.1371/journal.pgen.1005000.s020", "https://dx.doi.org/10.1371/journal.pgen.1005000.s021", "https://dx.doi.org/10.1371/journal.pgen.1005000.s022", "https://dx.doi.org/10.1371/journal.pgen.1005000.s023", "https://dx.doi.org/10.1371/journal.pgen.1005000.s024", "https://dx.doi.org/10.1371/journal.pgen.1005000.s025", "https://dx.doi.org/10.1371/journal.pgen.1005000.s026", "https://dx.doi.org/10.1371/journal.pgen.1005000.s027"], "stats"=>{"downloads"=>71, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transcriptome_Wide_Annotation_of_Eukaryotic_RNase_III_Reactivity_and_Degradation_Signals_/1322651", "title"=>"Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930701"], "description"=>"<p>(<b>A</b>) Bar graph indicating the percentage of known substrates (<b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s008\" target=\"_blank\">Table S1</a></b>) detected by each substrate detection assay. (<b>B</b>) Venn diagrams showing the number of Rnt1p cleavage targets identified by the different detection methods in protein-coding genes (top panel) or non-coding RNAs (bottom panel). (<b>C</b>) Box plots showing the distribution of the loop scores associated with the targets identified by each detection method. (<b>D</b>) Bar graph showing the percent of Rnt1p <i>in vitro</i> cleavage targets upregulated after the deletion of <i>RNT1</i> under different growth conditions. The RNA was extracted from <i>RNT1</i> and <i>rnt1∆</i> cells grown in media containing dextrose (2% Dex) or galactose (4% Gal) or cells shifted for 20 minutes in nitrogen supplemented media (20min N<sub>2</sub>). The expression levels were examined by quantitative RT-PCR using primers complementary to 109 <i>in vitro</i> cleavage targets not detected by the expression array (see <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s024\" target=\"_blank\">S17 Table</a></b>). Genes showing > 1.2 folds difference in expression between <i>rnt1∆</i> and <i>RNT1</i> cells with p-value < 0.01 were considered upregulated. The presented data are the average of three independent experiments. (<b>E</b>) Pie chart showing the percentage of Rnt1p cleavage sites associated with the accumulation of 5’-P cleavage products in <i>xrn1∆ / dcp2∆</i> cells [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref035\" target=\"_blank\">35</a>].</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322636, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g005", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Evaluation_of_the_different_methods_used_for_the_detection_of_Rnt1p_cleavage_targets_/1322636", "title"=>"Evaluation of the different methods used for the detection of Rnt1p cleavage targets.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930698"], "description"=>"<p>(<b>A</b>) Strategy for <u>S</u>equencing <u>A</u>ssisted <u>L</u>oop <u>I</u>dentification (SALI). In this strategy, <i>rnt1∆</i> RNA is cleaved by Rnt1p, RNA fragments < 150 nucleotides enriched and the 32–38 nucleotides internal cleavage fragments identified using next generation sequencing (NGS) (<b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s005\" target=\"_blank\">S5 Fig</a>.</b>). (<b>B</b>) Pie chart illustrating the types of RNAs associated with at least one enriched read cluster (details in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s022\" target=\"_blank\">S15 Table</a></b>). (<b>C</b>) Distribution of the top 30 enriched read clusters by RNA type (details in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s023\" target=\"_blank\">S16 Table</a></b>). (<b>D</b>) Cleavage of new substrates was verified using Northern blot as described in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.g001\" target=\"_blank\">Fig. 1G</a></b>. ACT1 mRNA and 5S rRNA were included as negative controls. The relative RNA expression (RMA) was calculated as described in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.g001\" target=\"_blank\">Fig. 1G</a></b>. (<b>E</b>) SALI uncovers new classes of Rnt1p substrates. The cleavages products identified by SALI were folded and the resulting structures shown in the form of pie chart. Unfolded RNAs and stems capped with loops larger than 6 nucleotides were classified as “other”. The loop sequence is described using standard single letter nucleotide code (IUPAC). (<b>F</b>) Validation of the new classes of Rnt1p substrates. A representative member of each loop type (unmodif) as well as mutated loop (MutL) and stem (MutS) versions were transcribed and cleaved by Rnt1p. The cleavage products were identified using 20% denaturating PAGE (see also <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s005\" target=\"_blank\">S5E Fig</a>.</b>). Substrate (S) and products (P) are indicated on the right. The size markers are indicated on the left. The observed cleavage sites are indicated by arrowheads.</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322633, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g004", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sequence_assisted_identification_of_Rnt1p_cleavage_signals_/1322633", "title"=>"Sequence assisted identification of Rnt1p cleavage signals.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930704"], "description"=>"<p>(<b>A</b>) Revised model of Rnt1p cleavage signals illustrating the common features of reactive RNAs. The substrates were classified based on the loop size and sequence and the three classes exhibiting statistically significant features (p-value < 0.05) were illustrated in the form of stem-loop structures. Underlined nucleotides indicate unpaired positions; black circles indicate paired positions. Grey underline, nucleotide or circle indicates the features, which are frequently observed, but not statistically enriched. Arrowheads indicate the cleavage sites. IBPB, BSB and CEB indicate initial binding and positioning box, binding stability box, and cleavage efficiency box [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref010\" target=\"_blank\">10</a>,<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref028\" target=\"_blank\">28</a>], respectively. The median Gibbs energy (∆G) was also calculated within each group of substrates. (<b>B</b>) The processing and degradation signals display different sequence preferences. The sequence of G2-loop substrates required for protein-coding mRNA degradation (MG2-loop; top panel) was compared to those involved in non-coding RNA processing (NCG2-loop; bottom panel) and the information content of each nucleotide position illustrated in the form of a composite bar graph. The shaded and underlined numbers indicate the position of the tetraloop and cleavage sites, respectively. (<b>C</b>) Rnt1p cleavage sites are preferentially base-paired in protein coding substrates. The percent paired nucleotides at each position of Rnt1p cleavage signals was determined in non-coding RNAs (ncRNA), protein-coding genes (PCGs) and randomly generated sequences (Random seq) and presented in the form of a line graph. Arrows indicate differences between the two groups of cleavage signals.</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322639, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g006", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Rnt1p_cleaves_different_classes_of_stem_loop_structure_with_diverse_sequence_and_structural_requirements_/1322639", "title"=>"Rnt1p cleaves different classes of stem-loop structure with diverse sequence and structural requirements.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930705"], "description"=>"<p>(<b>A</b>) RNA degradation signals accumulate in genes associated with carbohydrate metabolism and energy production. Genes affected by Rnt1p were classified using gene ontology (GO) [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref069\" target=\"_blank\">69</a>], MIPS database [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref039\" target=\"_blank\">39</a>] and literature search (<b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s025\" target=\"_blank\">S18</a> and <a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s026\" target=\"_blank\">S19 Table</a></b>) and those related to respiration and carbohydrate metabolism shown in the form of a bar graph. (<b>B</b>) The expression of Rnt1p substrates was examined in <i>RNT1</i> and <i>rnt1∆</i> cells grown on dextrose (2% Dex), galactose (4% Gal) or after different time following the shift from oxygen to nitrogen supplemented media (N<sub>2</sub>). The relative expression levels were determined using microarray (Expr. Array) or quantitative RT-PCR and presented in the form of heat-map (see also <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s006\" target=\"_blank\">S6 Fig</a>.</b>). Genes are grouped according to their response to oxygen depletion. The data shown are an average of three biological replicates. (<b>C</b>) <i>RNT1</i> and <i>rnt1∆</i> cells transformed with either the empty vector or pRS316 expressing the wild type <i>RNT1</i> allele (pRNT1) were stained with Rhodamine 123 (Rho123) and analyzed by flow cytometry. (<b>D</b>) <i>RNT1</i> and <i>rnt1∆</i> cells were stained using the Mitotracker stain (MTG) and analyzed by cytometry. (<b>E</b>) The ratio of the Rho123 to MTG signals were calculated for each strain and presented in the form of a bar graph. (<b>F</b>) <i>RNT1</i> (top) and <i>rnt1∆</i> (bottom) cells were stained with Mitotracker and the mitochondria (shown in green) visualized using epifluorescence. (<b>G</b>) Rnt1p is required for glycolytic oscillations. Glucose-depleted cell suspensions were supplemented with dextrose (Dex) and potassium cyanide (KCN) to induce oscillations and NADH fluorescence was recorded over time using a temperature-controlled spectrofluorometer.</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322640, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g007", "stats"=>{"downloads"=>0, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Deletion_of_RNT1_impairs_the_expression_of_in_vitro_substrates_associated_with_respiration_and_carbohydrate_metabolism_/1322640", "title"=>"Deletion of <i>RNT1</i> impairs the expression of <i>in vitro</i> substrates associated with respiration and carbohydrate metabolism.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930686"], "description"=>"<p>(<b>A</b>) Types of published Rnt1p substrates. Non-coding RNAs (ncRNAs) include snRNAs, snoRNAs and rRNA. Protein coding genes (PCGs) include ORFs, introns and UTRs (see <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s008\" target=\"_blank\">S1 Table</a></b>). (<b>B</b>) Schematic representation of the common features of Rnt1p G2-loop substrates. Unpaired nucleotides are underlined. Black circles indicate preferential nucleotide pairing. Enriched bases are named while N indicates any nucleotide. The cleavage sites are indicated by arrowheads. IBPB, BSB, MB and CEB indicate initial binding and positioning box, binding stability box, middle box and cleavage efficiency box [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref010\" target=\"_blank\">10</a>,<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref037\" target=\"_blank\">37</a>], respectively. (<b>C</b>) The score of known substrates was calculated (see <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s001\" target=\"_blank\">S1 Fig</a>.</b>) and displayed as histogram. (<b>D</b>) G2-loops were scored across the genome and the loop frequency indicated as histogram curve. (<b>E</b>) Pie chart illustrating the types of RNA associated with Rnt1p loops. Antisense and LTRs indicates orphan loops located opposite to an annotated gene or found in long terminal repeat elements, respectively. (<b>F</b>) Pie chart illustrating the types of RNA harboring the top 30 scoring loops (see <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s012\" target=\"_blank\">S5 Table</a></b>). (<b>G</b>) Northern blot analysis of Rnt1p cleavage products. RNA extracted from <i>RNT1</i> and <i>rnt1∆</i> cells was incubated with recombinant Rnt1p (<i>rnt1∆</i> + Rnt1p) and the cleavage products visualized using gene specific probes. ACT1 was used as loading control. The position of the rRNA and products (P) are indicated beside each gel. The relative RNA amount (RMA) was determined using quantitative RT-PCR and indicated below the gels.</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322621, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g001", "stats"=>{"downloads"=>0, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RNA_degradation_is_induced_by_context_dependent_activation_of_randomly_distributed_cleavage_motifs_/1322621", "title"=>"RNA degradation is induced by context dependent activation of randomly distributed cleavage motifs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/1930693"], "description"=>"<p>(<b>A</b>) Cut and Chip: a strategy for detecting Rnt1p substrates using tiling array. <i>rnt1∆</i> RNA was extracted, cleaved with recombinant Rnt1p and the cleavage products degraded using Xrn1p. Differences between the treated and untreated RNA was detected using Affymetrix tiling array and the cleavage fragments identified (<b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s004\" target=\"_blank\">S4 Fig</a>.</b>). (<b>B</b>) Cut and Chip accurately identifies Rnt1p targets. Comparison between the Mig2 mRNA degradation pattern [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref013\" target=\"_blank\">13</a>] detected by Cut and Chip (left panel) and Northern blot (right panel). ACT1 mRNA was included as negative control [<a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.ref013\" target=\"_blank\">13</a>]. The relative levels of RNA detected by the different probes (black dots) are presented below each ORF. The cleavage sites are shown as stem-loops and the cleaved segments indicated by black line. The Northern blot was performed as described in <b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.g001\" target=\"_blank\">Fig. 1G</a></b>. (<b>C</b>) Pie chart illustrating the types of RNA cleaved by Rnt1p (<b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s018\" target=\"_blank\">S11 Table</a></b>). (<b>D</b>) Pie chart illustrating the type of the top 30 cleaved RNAs (<b><a href=\"http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005000#pgen.1005000.s019\" target=\"_blank\">S12 Table</a></b>). (<b>E</b>) Identification of Rnt1p cleavage site using primer extension. Reverse transcription was performed using gene specific radiolabelled primers before (<i>rnt1</i>∆) and after (<i>rnt1</i>∆ + Rnt1p) cleavage. NT, M and P indicate no template control, size markers, and the cleavage product 5’ end, respectively. The smoothed cleavage profile is shown relative to the ORF on top. The predicted G2-loops are indicated as stem loops and the detected cleavage site (CS) identified by the arrows. Probes used for primer extension are shown below each ORF.</p>", "links"=>[], "tags"=>["Transcriptome Wide Annotation", "sequence", "Rnt 1p reactivity", "impact", "Eukaryotic RNase III Reactivity", "growth conditions", "RNA degradation signals", "transcriptome stability", "gene", "Rnt 1p cleavage signals", "Degradation Signals Detection", "expression"], "article_id"=>1322628, "categories"=>["Biological Sciences"], "users"=>["Jules Gagnon", "Mathieu Lavoie", "Mathieu Catala", "Francis Malenfant", "Sherif Abou Elela"], "doi"=>"https://dx.doi.org/10.1371/journal.pgen.1005000.g003", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gene_expression_independent_identification_of_RNA_degradation_targets_/1322628", "title"=>"Gene expression independent identification of RNA degradation targets.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-13 17:38:45"}

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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