The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot
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Mendeley | Further Information

{"title"=>"The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot", "type"=>"journal", "authors"=>[{"first_name"=>"Richa", "last_name"=>"Sardana", "scopus_author_id"=>"24175073600"}, {"first_name"=>"Xin", "last_name"=>"Liu", "scopus_author_id"=>"55866149400"}, {"first_name"=>"Sander", "last_name"=>"Granneman", "scopus_author_id"=>"6602171904"}, {"first_name"=>"Jieyi", "last_name"=>"Zhu", "scopus_author_id"=>"56177875500"}, {"first_name"=>"Michael", "last_name"=>"Gill", "scopus_author_id"=>"56177515100"}, {"first_name"=>"Ophelia", "last_name"=>"Papoulas", "scopus_author_id"=>"6602206323"}, {"first_name"=>"Edward M.", "last_name"=>"Marcotte", "scopus_author_id"=>"7003412942"}, {"first_name"=>"David", "last_name"=>"Tollervey", "scopus_author_id"=>"7005480769"}, {"first_name"=>"Carl C.", "last_name"=>"Correll", "scopus_author_id"=>"7003584105"}, {"first_name"=>"Arlen W.", "last_name"=>"Johnson", "scopus_author_id"=>"7410015390"}], "year"=>2015, "source"=>"PLoS Biology", "identifiers"=>{"issn"=>"15457885", "pui"=>"607875719", "doi"=>"10.1371/journal.pbio.1002083", "sgr"=>"84942135052", "scopus"=>"2-s2.0-84942135052", "isbn"=>"1545-7885 (Electronic)\r1544-9173 (Linking)", "pmid"=>"25710520"}, "id"=>"ac46fc42-7dca-3d37-ab8b-4f2091b78f1c", "abstract"=>"In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.", "link"=>"http://www.mendeley.com/research/deahbox-helicase-dhr1-dissociates-u3-prerrna-promote-formation-central-pseudoknot", "reader_count"=>35, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Student > Doctoral Student"=>5, "Researcher"=>4, "Student > Ph. D. Student"=>13, "Student > Master"=>1, "Student > Bachelor"=>4, "Lecturer"=>1, "Professor"=>4}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Student > Doctoral Student"=>5, "Researcher"=>4, "Student > Ph. D. Student"=>13, "Student > Master"=>1, "Student > Bachelor"=>4, "Lecturer"=>1, "Professor"=>4}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>14, "Medicine and Dentistry"=>1, "Agricultural and Biological Sciences"=>17, "Chemistry"=>1, "Psychology"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>17}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>14}}, "reader_count_by_country"=>{"United States"=>1, "United Kingdom"=>1, "Switzerland"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1922760"], "description"=>"<p>U3 binds to the pre-rRNA cotranscriptionally. Binding to the 5′-end of 18S and the downstream element (nts 1139–1142) brings together the elements of the CPK. Following cleavage at A0 and A1, U3 is displaced to allow folding of the CPK and cleavage at A2 to liberate the pre-40S. Watson-Crick base pairs (|), non-Watson base pairs (*).</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316905, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g001", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_transition_from_the_pre_ribosome_to_the_pre_40S_/1316905", "title"=>"The transition from the pre-ribosome to the pre-40S.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922761"], "description"=>"<p>(A–D) Whole cell extracts were prepared from cycloheximide-treated cells expressing WT Dhr1–13myc (pAJ2311) (upper panels) or <i>dhr1-K420A</i> (pAJ3081) (lower panels) in yeast strain AJY3711 (<i>P</i><sub><i>GAL</i></sub>-<i>3xHA-DHR1</i>) shifted to glucose media for 6 h to deplete genomically expressed 3xHA-Dhr1. (A, B) Extracts were subjected to sucrose density gradient ultracentrifugation and absorbance at 254 nm was monitored continuously throughout the gradients. Additional supporting data are provided in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s001\" target=\"_blank\">S1 Data</a>. (C, D) Proteins were precipitated from fractions and subjected to SDS-PAGE followed by Western blotting. Dhr1–13myc, Mpp10, Imp4, Rps8, and Rpl8 were detected using anti-myc, anti-Mpp10, anti-Imp4, anti-Rps8, and anti-Rpl8 antibodies, respectively. The positions of 40S, 60S, and 80S, determined by monitoring absorbance at 254 nm, are indicated. (E) RNA was prepared from gradients as described for (A, B) and separated by agarose/formaldehyde gel electrophoresis. U3 was detected by Northern blotting using oligo AJO1686.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316906, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g002", "stats"=>{"downloads"=>2, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dhr1_K420A_accumulates_a_novel_8764_45S_particle_containing_SSU_components_/1316906", "title"=>"Dhr1<sub>K420A</sub> accumulates a novel ∼45S particle containing SSU components.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922768"], "description"=>"<p>(A) Cultures of AJY3711 (<i>P</i><sub><i>GAL</i></sub>-<i>3xHA-DHR1</i>) expressing untagged WT Dhr1 (pAJ3082), WT Dhr1–13myc (pAJ2311), or <i>dhr1</i><sub><i>K420A</i></sub>-<i>13myc</i> (pAJ3081) were shifted to glucose media for 6 h to deplete 3xHA-Dhr1. RNA was prepared from whole cell extracts (Input) or immunoprecipitated samples (IP) and separated by electrophoresis through agarose/formaldehyde gels or denaturing polyacrylamide gels for the A0-A1 fragment and U3. RNAs were detected by Northern blotting using probes specific to A2-A3 (AJO603), D-A2 (AJO130), A0-A1 (AJO1850), and U3 (AJO1686). (B) Table of proteins identified by MS in the Dhr1<sub>K420A</sub> particle. Only proteins with at least three peptide-spectrum matches are listed. (C) The CPK (red), 18S rRNA (cyan), and r-proteins identified in the Dhr1<sub>K420A</sub> particle (3B) are shown in orange in the structure of the mature <i>S</i>. <i>cerevisiae</i> 40S subunit (left). For comparison the proteins missing from the Dhr1<sub>K420A</sub> particle are shown in yellow on the right. The Dhr1<sub>K420A</sub> particle likely adopts a more open conformation in the absence of r-proteins.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316908, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g003", "stats"=>{"downloads"=>1, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dhr1_K420A_co_immunoprecipitates_U3_and_associated_proteins_and_20S_and_21S_pre_rRNAs_/1316908", "title"=>"Dhr1<sub>K420A</sub> co-immunoprecipitates U3 and associated proteins and 20S and 21S pre-rRNAs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922770"], "description"=>"<p>(A) Cartoon showing expected U3–18S rRNA base-pairing and the same region of 18S rRNA in the CPK of the mature 40S subunit, based on crystal structure (PDB 3U5B). (B) DMS was used to probe accessibility of A1139. Extracts were prepared from cells expressing WT Dhr1 or Dhr1<sub>K420A</sub>, as described in the legend to <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.g002\" target=\"_blank\">Fig. 2</a> and fractionated on sucrose density gradients. The 45S regions of the gradients were harvested and treated with DMS as indicated. DMS modification was detected by primer extension using radiolabeled primer AJO1849. Peak areas were quantified using ImageJ (NIH). Purified mature 40S subunits were used as a control and a sequencing ladder was generated using a DNA template containing the 18S rDNA gene.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316910, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g004", "stats"=>{"downloads"=>7, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U3_remains_base_paired_with_18S_rRNA_in_a_dhr1_mutant_/1316910", "title"=>"U3 remains base-paired with 18S rRNA in a <i>dhr1</i> mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922808", "https://ndownloader.figshare.com/files/1922809", "https://ndownloader.figshare.com/files/1922810", "https://ndownloader.figshare.com/files/1922811", "https://ndownloader.figshare.com/files/1922812", "https://ndownloader.figshare.com/files/1922813", "https://ndownloader.figshare.com/files/1922814", "https://ndownloader.figshare.com/files/1922815", "https://ndownloader.figshare.com/files/1922816", "https://ndownloader.figshare.com/files/1922817", "https://ndownloader.figshare.com/files/1922818", "https://ndownloader.figshare.com/files/1922819", "https://ndownloader.figshare.com/files/1922820", "https://ndownloader.figshare.com/files/1922821", "https://ndownloader.figshare.com/files/1922822", "https://ndownloader.figshare.com/files/1922823", "https://ndownloader.figshare.com/files/1922824", "https://ndownloader.figshare.com/files/1922825", "https://ndownloader.figshare.com/files/1922826"], "description"=>"<div><p>In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked <i>in vivo</i> to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an <i>in vivo</i> substrate of Dhr1. To support the conclusions derived from <i>in vivo</i> analysis we showed that Dhr1 unwinds U3-18S duplexes <i>in vitro</i> by using a mechanism reminiscent of DEAD box proteins.</p></div>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316944, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.1002083.s001", "https://dx.doi.org/10.1371/journal.pbio.1002083.s002", "https://dx.doi.org/10.1371/journal.pbio.1002083.s003", "https://dx.doi.org/10.1371/journal.pbio.1002083.s004", "https://dx.doi.org/10.1371/journal.pbio.1002083.s005", "https://dx.doi.org/10.1371/journal.pbio.1002083.s006", "https://dx.doi.org/10.1371/journal.pbio.1002083.s007", "https://dx.doi.org/10.1371/journal.pbio.1002083.s008", "https://dx.doi.org/10.1371/journal.pbio.1002083.s009", "https://dx.doi.org/10.1371/journal.pbio.1002083.s010", "https://dx.doi.org/10.1371/journal.pbio.1002083.s011", "https://dx.doi.org/10.1371/journal.pbio.1002083.s012", "https://dx.doi.org/10.1371/journal.pbio.1002083.s013", "https://dx.doi.org/10.1371/journal.pbio.1002083.s014", "https://dx.doi.org/10.1371/journal.pbio.1002083.s015", "https://dx.doi.org/10.1371/journal.pbio.1002083.s016", "https://dx.doi.org/10.1371/journal.pbio.1002083.s017", "https://dx.doi.org/10.1371/journal.pbio.1002083.s018", "https://dx.doi.org/10.1371/journal.pbio.1002083.s019"], "stats"=>{"downloads"=>66, "page_views"=>40, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_DEAH_box_Helicase_Dhr1_Dissociates_U3_from_the_Pre_rRNA_to_Promote_Formation_of_the_Central_Pseudoknot_/1316944", "title"=>"The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922774"], "description"=>"<p>(A) The parental and a Dhr1-HTP tagged strain were subjected to the CRAC protocol (see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#sec015\" target=\"_blank\">Materials and Methods</a>), cross-linked RNA was partially digested, radioactively labeled, ligated to linkers, and after nickel purification resolved on a 4%–12% NuPAGE gel. Protein-RNA complex was transferred to nitrocellulose and RNA was extracted from the regions indicated by a red dashed box. (B) Dhr1 preferentially cross-links to U3. Reads from Dhr1 (<i>n</i> = 2) and a negative control CRAC experiment were mapped to the 2008 <i>S</i>. <i>cerevisiae</i> genomic reference sequence and mapped reads were assigned to genomic features. The histogram shows the average percentage of all mapped reads that contained box C/D and (C) box H/ACA snoRNA sequences. Note that only a very small fraction of the reads from the control experiment mapped to snoRNAs. (D) Dhr1 preferentially cross-links to the 5′ end of the U3. Plotted is the average read distribution frequency over the U3A (snR17A) gene generated from two Dhr1 CRAC datasets. A schematic representation of the U3 gene and functional sequence elements are indicated below the plot. (E) Same as in (D) but for nucleotide substitutions. The secondary structure of the U3 was adopted from Granneman and colleagues [<a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.ref031\" target=\"_blank\">31</a>] and generated using VARNA (<a href=\"http://varna.lri.fr\" target=\"_blank\">http://varna.lri.fr</a>). The coloring indicates the frequency by which the nucleotide was substituted. Additional supporting data are provided in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s002\" target=\"_blank\">S2 Data</a>.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316914, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g005", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Dhr1_cross_links_to_U3_/1316914", "title"=>"Dhr1 cross-links to U3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922775"], "description"=>"<p>(A) Random mutations were introduced the entire coding region of U3 (<i>SNR17A</i>) by low fidelity PCR. The mutant PCR library was then recombined into an expression vector <i>in vivo</i> in AJY3752 (<i>P</i><sub><i>GAL</i></sub>-<i>DHR1 P</i><sub><i>GAL</i></sub>-<i>SNR17A snr17B∆</i>) expressing <i>dhr1-cs2</i> (pAJ3095) and transformants were screened for improved growth at 20°C. Individual mutations from suppressing clones were subcloned and their ability to suppress <i>dhr1-cs2</i> was compared by serial dilution assay. Top row, WT <i>DHR1</i> with WT U3; second row, <i>dhr1-cs2</i> with WT U3 as controls. Plates were incubated at 20°C for 7 days. (B) The positions of U3 mutations that suppress <i>dhr1-cs2</i> and residues most frequently cross-linked to Dhr1, identified by substitutions in the CRAC analysis, are mapped to the predicted U3–18S secondary structure.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316915, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g006", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mutations_in_the_5_8242_end_of_U3_suppress_a_cold_sensitive_dhr1_mutant_/1316915", "title"=>"Mutations in the 5′-end of U3 suppress a cold-sensitive dhr1 mutant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922776"], "description"=>"<p>(A) Initial velocities of P<sub>i</sub> released after addition of 1 mM ATP at room temperature (RT) in the presence or absence of the indicated RNA with either WT Dhr1, Dhr1<sub>D516A/E517A</sub>, Dhr1<sub>K420A</sub>, or mock. Activity of Dhr1 either in the presence or absence of Imp3 was also determined. (B) Schematic of the putative ATP binding site of Dhr1 with expected contacts from K420 (motif I) and from D516 and E517 (motif II). (C) Determination of the ATP <i>k</i><sub>cat</sub> and <i>K</i><sub>m</sub> for Dhr1 with the Michaelis-Menten plots shown in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s014\" target=\"_blank\">S7 Fig</a>. Additional supporting data are provided in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s003\" target=\"_blank\">S3</a> and <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s004\" target=\"_blank\">S4 Data</a>.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316916, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g007", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_vitro_ATPase_activity_of_Dhr1_/1316916", "title"=>"<i>In vitro</i> ATPase activity of Dhr1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922778"], "description"=>"<p>(A) Cartoon of U3-ETS2 substrate used for unwinding assays. Full length U3 snoRNA was used for the reactions in (B, C, and D). A truncated version of U3 snoRNA (nts 1–76) was used in the reactions in (E and F) to avoid non-specific interaction between Dhr1 and the 3′ region of U3 snoRNA. (B) Reaction scheme for the pre-steady state conditions ([E] > [duplex]) with results in (C and D). (C) Representative unwinding reactions stopped after 20 min. Electrophoretic mobility shift assay (EMSA) separated the <sup>32</sup>P-labeled ETS2 free (unwound) from its duplex form. The RT reaction contained 500 nM Dhr1, 1 mM ATP, ≤0.3 nM U3-ET2 duplex with other reagents described in Materials and Methods. (D) Fraction unwound is plotted as a function of time: after addition of either ATP in the presence of either Dhr1 (purple circle), Dhr1<sub>D516A/E517A</sub> (blue square), Dhr1<sub>K420A</sub> (green diamond); after addition of ADP in the presence of either Dhr1 (open purple circle) or Dhr1<sub>D516A/E517A</sub> (open blue square); or after addition of Dhr1 (circle with a cross). (E) Outline of the turnover of excess substrate reaction scheme under steady state conditions ([duplex] > [E]) with results in (F). The reaction contained 100 nM Dhr1, 6 mM ATP, 500 nM U3-ETS2 radiolabeled duplex, and 5 μM of unlabeled ETS2 with other reagents described in Materials and Methods. (F) Fraction unwound is plotted as a function of time with symbols as in (D). Additional supporting data are provided in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s005\" target=\"_blank\">S5</a> and <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s006\" target=\"_blank\">S6 Data</a>.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316918, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g008", "stats"=>{"downloads"=>2, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U3_ETS2_duplex_unwinding_by_Dhr1_/1316918", "title"=>"U3-ETS2 duplex unwinding by Dhr1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/1922779"], "description"=>"<p>(A) Reaction schematic for annealing the U3–18S duplex by Imp3 and duplex unwinding by Dhr1. (B) Reaction scheme for the pre-steady state conditions ([E] > [duplex]) with results shown in (C and D). (C) Representative RT unwinding single time point data after 60 min. From EMSA showing <sup>32</sup>P-labeled 18S free (unwound) or in duplex with U3 MINI in a complex with Imp3 using 500 nM Dhr1, 1 mM ATP, ≤1 nM U3–18S duplex and other reagents described in Materials and Methods. (D) Time-dependent reactions with symbols as defined in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.g008\" target=\"_blank\">Fig. 8D</a>. Additional supporting data are provided in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002083#pbio.1002083.s007\" target=\"_blank\">S7 Data</a>.</p>", "links"=>[], "tags"=>["cpk", "DEAD box proteins", "U 3 sequences", "U 3 box", "region", "displacing U 3.", "vivo", "rna", "U 3", "Dhr 1"], "article_id"=>1316919, "categories"=>["Uncategorised"], "users"=>["Richa Sardana", "Xin Liu", "Sander Granneman", "Jieyi Zhu", "Michael Gill", "Ophelia Papoulas", "Edward M. Marcotte", "David Tollervey", "Carl C. Correll", "Arlen W. Johnson"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1002083.g009", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_U3_8211_18S_duplex_unwinding_by_Dhr1_/1316919", "title"=>"U3–18S duplex unwinding by Dhr1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-24 03:09:17"}

PMC Usage Stats | Further Information

  • {"unique-ip"=>"12", "full-text"=>"11", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"19", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"8"}
  • {"unique-ip"=>"10", "full-text"=>"7", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"7", "cited-by"=>"0", "year"=>"2016", "month"=>"9"}
  • {"unique-ip"=>"7", "full-text"=>"10", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2016", "month"=>"10"}
  • {"unique-ip"=>"10", "full-text"=>"8", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2016", "month"=>"11"}
  • {"unique-ip"=>"6", "full-text"=>"4", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2016", "month"=>"12"}
  • {"unique-ip"=>"10", "full-text"=>"10", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2017", "month"=>"1"}
  • {"unique-ip"=>"8", "full-text"=>"8", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"2"}
  • {"unique-ip"=>"6", "full-text"=>"6", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"3"}
  • {"unique-ip"=>"2", "full-text"=>"2", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"4"}
  • {"unique-ip"=>"7", "full-text"=>"11", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"5"}
  • {"unique-ip"=>"7", "full-text"=>"5", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"6"}
  • {"unique-ip"=>"11", "full-text"=>"12", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2017", "month"=>"7"}
  • {"unique-ip"=>"6", "full-text"=>"7", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"8"}
  • {"unique-ip"=>"11", "full-text"=>"9", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"7", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
  • {"unique-ip"=>"29", "full-text"=>"11", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"17", "cited-by"=>"1", "year"=>"2017", "month"=>"10"}
  • {"unique-ip"=>"12", "full-text"=>"13", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"11"}
  • {"unique-ip"=>"5", "full-text"=>"7", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
  • {"unique-ip"=>"6", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"10", "supp-data"=>"3", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
  • {"unique-ip"=>"7", "full-text"=>"6", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"3"}
  • {"unique-ip"=>"6", "full-text"=>"4", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"6", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"1"}
  • {"unique-ip"=>"10", "full-text"=>"9", "pdf"=>"6", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"9", "full-text"=>"11", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"13", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2018", "month"=>"5"}
  • {"unique-ip"=>"7", "full-text"=>"8", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"11", "full-text"=>"8", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"5", "full-text"=>"3", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"7", "full-text"=>"7", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"4", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"10", "full-text"=>"10", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"13", "full-text"=>"9", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"6", "full-text"=>"5", "pdf"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"7", "full-text"=>"5", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"8", "full-text"=>"19", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"10", "full-text"=>"7", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}

Relative Metric

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