Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein
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{"title"=>"Normal microRNA maturation and germ-line stem cell maintenance requires loquacious, a double-stranded RNA-binding domain protein", "type"=>"journal", "authors"=>[{"first_name"=>"Klaus", "last_name"=>"Förstemann", "scopus_author_id"=>"17342159500"}, {"first_name"=>"Yukihide", "last_name"=>"Tomari", "scopus_author_id"=>"6603810250"}, {"first_name"=>"Tingting", "last_name"=>"Du", "scopus_author_id"=>"23667159500"}, {"first_name"=>"Vasily V.", "last_name"=>"Vagin", "scopus_author_id"=>"12782815200"}, {"first_name"=>"Ahmet M.", "last_name"=>"Denli", "scopus_author_id"=>"6506096312"}, {"first_name"=>"Diana P.", "last_name"=>"Bratu", "scopus_author_id"=>"6603915694"}, {"first_name"=>"Carla", "last_name"=>"Klattenhoff", "scopus_author_id"=>"8665927900"}, {"first_name"=>"William E.", "last_name"=>"Theurkauf", "scopus_author_id"=>"7003327650"}, {"first_name"=>"Phillip D.", "last_name"=>"Zamore", "scopus_author_id"=>"7003593294"}], "year"=>2005, "source"=>"PLoS Biology", "identifiers"=>{"isbn"=>"1545-7885 (Electronic)\\r1544-9173 (Linking)", "scopus"=>"2-s2.0-22744433240", "pui"=>"41032187", "doi"=>"10.1371/journal.pbio.0030236", "sgr"=>"22744433240", "pmid"=>"15918770", "issn"=>"15449173"}, "id"=>"5cbac970-fc69-35fc-a02a-8911f1f2056d", "abstract"=>"microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs(f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.", "link"=>"http://www.mendeley.com/research/normal-microrna-maturation-germline-stem-cell-maintenance-requires-loquacious-doublestranded-rnabind", "reader_count"=>207, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>22, "Researcher"=>51, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>60, "Student > Postgraduate"=>8, "Student > Master"=>24, "Other"=>4, "Student > Bachelor"=>13, "Lecturer"=>3, "Lecturer > Senior Lecturer"=>2, "Professor"=>16}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>22, "Researcher"=>51, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>60, "Student > Postgraduate"=>8, "Student > Master"=>24, "Other"=>4, "Student > Bachelor"=>13, "Lecturer"=>3, "Lecturer > Senior Lecturer"=>2, "Professor"=>16}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>25, "Materials Science"=>1, "Agricultural and Biological Sciences"=>157, "Medicine and Dentistry"=>10, "Neuroscience"=>2, "Sports and Recreations"=>1, "Physics and Astronomy"=>1, "Chemistry"=>2, "Computer Science"=>5, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>10}, "Neuroscience"=>{"Neuroscience"=>2}, "Chemistry"=>{"Chemistry"=>2}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>157}, "Computer Science"=>{"Computer Science"=>5}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>25}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"Hungary"=>1, "United States"=>11, "United Kingdom"=>2, "Russia"=>1, "Spain"=>1, "Canada"=>1, "Belgium"=>1, "China"=>2, "Denmark"=>1, "Brazil"=>1, "South Africa"=>1, "Mexico"=>1, "France"=>2}, "group_count"=>9}

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  • {"month"=>"5", "year"=>"2016", "pdf_views"=>"3", "xml_views"=>"0", "html_views"=>"60"}
  • {"month"=>"6", "year"=>"2016", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"20"}
  • {"month"=>"7", "year"=>"2016", "pdf_views"=>"6", "xml_views"=>"0", "html_views"=>"41"}
  • {"month"=>"8", "year"=>"2016", "pdf_views"=>"12", "xml_views"=>"0", "html_views"=>"53"}
  • {"month"=>"9", "year"=>"2016", "pdf_views"=>"13", "xml_views"=>"0", "html_views"=>"52"}
  • {"month"=>"10", "year"=>"2016", "pdf_views"=>"13", "xml_views"=>"0", "html_views"=>"33"}
  • {"month"=>"11", "year"=>"2016", "pdf_views"=>"4", "xml_views"=>"0", "html_views"=>"38"}
  • {"month"=>"12", "year"=>"2016", "pdf_views"=>"10", "xml_views"=>"0", "html_views"=>"37"}
  • {"month"=>"1", "year"=>"2017", "pdf_views"=>"12", "xml_views"=>"0", "html_views"=>"38"}
  • {"month"=>"2", "year"=>"2017", "pdf_views"=>"12", "xml_views"=>"1", "html_views"=>"34"}
  • {"month"=>"3", "year"=>"2017", "pdf_views"=>"15", "xml_views"=>"0", "html_views"=>"113"}
  • {"month"=>"4", "year"=>"2017", "pdf_views"=>"5", "xml_views"=>"0", "html_views"=>"80"}
  • {"month"=>"5", "year"=>"2017", "pdf_views"=>"11", "xml_views"=>"7", "html_views"=>"42"}
  • {"month"=>"6", "year"=>"2017", "pdf_views"=>"0", "xml_views"=>"1", "html_views"=>"22"}
  • {"month"=>"7", "year"=>"2017", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"33"}
  • {"month"=>"8", "year"=>"2017", "pdf_views"=>"4", "xml_views"=>"1", "html_views"=>"19"}
  • {"month"=>"9", "year"=>"2017", "pdf_views"=>"9", "xml_views"=>"1", "html_views"=>"26"}
  • {"month"=>"10", "year"=>"2017", "pdf_views"=>"7", "xml_views"=>"1", "html_views"=>"36"}
  • {"month"=>"11", "year"=>"2017", "pdf_views"=>"7", "xml_views"=>"0", "html_views"=>"27"}

Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/962574"], "description"=>"<div><p>(A) Pre-miRNA processing activity co-immunoprecipitates with myc-tagged Loqs PB and with endogenous Dcr-1 or endogenous Loqs, but not with myc-tagged GFP.</p>\n <p>(B) Pre-miRNA processing activity co-purifies by immunoprecipitation with both Loqs protein isoforms that interact with Dcr-1, Loqs PA, and Loqs PB. The extracts used in (A) and (B) were independently prepared.</p></div>", "links"=>[], "tags"=>["pre-mirna", "s2"], "article_id"=>632546, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g005", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loqs_Is_Associated_with_Pre_miRNA_Processing_Activity_in_S2_Cells_/632546", "title"=>"Loqs Is Associated with Pre-miRNA Processing Activity in S2 Cells", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:05:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/962440"], "description"=>"<div><p>(A) Dcr-1 associates with myc-tagged Loqs PA or PB, and with endogenous Loqs protein. Immunoprecipitation with anti-myc or anti-Loqs antibody was performed using lysates from S2 cells transfected with the indicated expression plasmid. Dcr-1 was detected by Western blotting.</p>\n <p>(B) myc-tagged Loqs PB stably associates with Dcr-1 but not Dcr-2. S2 cells were transfected with plasmid expressing myc-tagged Loqs PB, then lysed and immunoprecipitated with anti-myc antibody. The immunoprecipitates were analyzed by Western blotting using anti-Dcr-1 or anti-Dcr-2 antibodies.</p>\n <p>(C) S2 cells were transfected with plasmid expressing myc-tagged GFP, Loqs PA, or Loqs PB, then extracted and immunoprecipitated with anti-Dcr-1 antibody. The immunoprecipitates were analyzed by Western blotting using anti-myc antibody.</p>\n <p>(D) Anti-Dcr-1 antibody was used to immunoprecipitate Dcr-1 and associated proteins from S2 cell lysates, and the immunoprecipitates were analyzed by Western blotting using anti-Loqs antibody to detect endogenous Loqs protein. The major Loqs protein isoform recovered was Loqs PB. In a longer exposure (bottom panel), a band corresponding in size to Loqs PA is visible. The most abundant Loqs isoform the input sample, Loqs PC, which lacks the third dsRBD, did not immunoprecipitate with Dcr-1, suggesting that the third dsRBD is required for the association of Loqs with Dcr-1.</p></div>", "links"=>[], "tags"=>["dcr-1"], "article_id"=>632420, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g004", "stats"=>{"downloads"=>8, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loqs_and_Dcr_1_Are_Present_in_a_Common_Protein_Complex_in_S2_Cells_/632420", "title"=>"Loqs and Dcr-1 Are Present in a Common Protein Complex in S2-Cells", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:04:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/962380"], "description"=>"<div><p>(A) <i>loqs</i><sup>f00791</sup> mutant ovary lysates processed pre-<i>let-7</i> into mature <i>let-7</i> miRNA ∼19-fold more slowly than wild-type. The data were fit to a first-order exponential equation, and initial velocities calculated from the fitted curve.</p>\n <p>(B) Analysis of pre-<i>let-7</i> processing in extracts from S2 cells. The cells were treated twice with dsRNA corresponding to the indicated genes.</p></div>", "links"=>[], "tags"=>["Computational biology", "cell biology", "developmental biology", "genetics and genomics", "molecular biology", "Biochemistry"], "article_id"=>632357, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g003", "stats"=>{"downloads"=>2, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loqs_Is_Required_for_Efficient_pre_let_7_Processing_In_Vitro_/632357", "title"=>"Loqs Is Required for Efficient pre-<i>let</i>-<i>7</i> Processing In Vitro", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:03:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/962674"], "description"=>"<div><p>(A) S2 cell lysate was fractionated by gel filtration chromatography and analyzed for pre-<i>let-7</i> processing activity, and Dcr-1, Dcr-2, and Loqs proteins.</p>\n <p>(B) The sizes of the distinct complexes containing Loqs (∼630 kDa), Dcr-1 (∼480 kDa), and Dcr-2 (∼230 kDa) and the broad complex containing pre-miRNA processing activity (∼525 kDa) were estimated using molecular weight standards (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; chymotrypsinogen A, 25 kDa) and recombinant Dcr-2 and R2D2 proteins (rDcr-2 and rR2D2). The blue asterisk denotes the peak of pre-<i>let-7</i> processing activity detected in (A).</p>\n <p>(C) Fractions containing the Dcr-1 peak were pooled and immunoprecipitated with either anti-Dcr-1 or anti-Loqs antibodies. Western blotting with anti-Dcr-1 and anti-Loqs antibodies demonstrated that Dcr-1 and Loqs remained associated through gel filtration chromatography.</p></div>", "links"=>[], "tags"=>["complexes", "containing", "pre-mirna"], "article_id"=>632646, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g006", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_Complexes_Containing_Pre_miRNA_Processing_Activity_Dcr_1_and_Loqs_/632646", "title"=>"Analysis of Complexes Containing Pre-miRNA Processing Activity, Dcr-1, and Loqs", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:06:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/473159", "https://ndownloader.figshare.com/files/473203"], "description"=>"<div><p>microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of <em>Drosophila</em> pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV <em>trans</em>-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of <em>white</em> by a dsRNA trigger, and silencing of the endogenous <em>Stellate</em> locus by <em>Suppressor of Stellate,</em> all require Loqs. In <em>loqs</em><sup>f00791</sup> mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the <em>Drosophila</em> RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known <em>Drosophila</em> RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis.</p> </div>", "links"=>[], "tags"=>["microrna", "maturation", "germ-line", "requires", "double-stranded", "rna-binding"], "article_id"=>153224, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.0030236.sg001", "https://dx.doi.org/10.1371/journal.pbio.0030236.sg002"], "stats"=>{"downloads"=>12, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Normal_microRNA_Maturation_and_Germ_Line_Stem_Cell_Maintenance_Requires_Loquacious_a_Double_Stranded_RNA_Binding_Domain_Protein/153224", "title"=>"Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-01-20 11:31:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/963116"], "description"=>"<div><p>(A) Wild-type ovarioles contain a germarium and a developmentally ordered array of six to eight egg chambers, whereas <i>loqs</i><sup>f00791</sup> mutant ovarioles contain a smaller than normal germarium, two or three pre-vitellogenic egg chambers, and a late-stage egg chamber. Wild-type and <i>loqs</i> ovarioles are shown at the same magnification.</p>\n <p>(B) In wild-type ovarioles, the germarium contains several newly formed germ-line cysts surrounded by somatic follicle cells. In contrast, <i>loqs</i><sup>f00791</sup> mutant germaria contain few germ-line cells, which are not organized into distinct cysts. The follicle cell layer is also significantly reduced in<i>loqs</i><sup>f00791</sup> germaria.</p>\n <p>(C) Wild-type and <i>loqs</i> mutant germaria labeled for α-Spectrin (green) and filamentous Actin (red). In wild type, anti-α-Spectrin labels the spectrosome (ss), a structure characteristic of germ-line stem cells, which are normally found at the anterior of the germarium, apposed to the somatic terminal cells (tc). The cystoblasts, the daughters of the stem cells, also contain a spectrosome, but are located posterior to the stem cells. In <i>loqs</i> mutant ovaries, spectrosome-containing cells were not detected, indicating that normal germ-line stem cells are not present. These observations indicate that stem cells are not maintained.</p>\n <p>In (A) and (B), ovaries were labeled for filamentous actin (red) using rhodamine phalloidin, DNA (blue) using TOTO3 (Molecular Probes), and the germ-line marker Vasa (green) using rabbit anti-Vasa antibody detected with fluorescein-conjugated anti-rabbit secondary antibody. In (B) and (C), wild-type and <i>loqs</i> germaria are shown at the same magnification.</p></div>", "links"=>[], "tags"=>["germ-line"], "article_id"=>633067, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g010", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_loqs_f00791_Fail_to_Maintain_Germ_Line_Stem_Cells_/633067", "title"=>"loqs<sup>f00791</sup> Fail to Maintain Germ-Line Stem Cells", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:09:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/962249"], "description"=>"<div><p>(A) Northern analysis of total RNA from wild-type, <i>loqs</i><sup>f00791</sup> heterozygotes and homozygotes, and <i>r2d2</i> heterozygotes and homozygotes for whole males, probed for miR-277 and <i>bantam</i>. The membrane was first hybridized with the miR-277 probe, stripped and probed for 2S rRNA as a loading control, then stripped again and probed for <i>bantam</i> miRNA. Asterisk: the 2S probe was not completely removed before the hybridization with the <i>bantam</i> probe, resulting in an additional band above the mature <i>bantam</i> RNA.</p>\n <p>(B) Total RNA from whole males, female carcasses remaining after ovary dissection, and dissected ovaries was probed for miR-7. As a control for successful dissection, the blot was also probed for miR-277, which is not expressed in ovaries (KF and PDZ, unpublished results). 2S rRNA again served as a loading control.</p>\n <p>(C) Depletion of <i>dcr-1</i> or <i>loqs</i> in S2 cells by RNAi leads to pre-miRNA accumulation. Total RNA was isolated after dsRNA-triggered RNAi of the indicated genes. The control sample was treated with dsRNA corresponding to the polylinker sequence of pLitmus28i.</p>\n <p>(D) Depletion of Dcr-1, Dcr-2, Loqs, and Drosha was confirmed by Western blotting.</p>\n <p>(E) Western blotting analysis demonstrates that Dcr-1 levels are not significantly reduced by depletion of Loqs by RNAi in S2 cells, but are lower in <i>loqs</i><sup>f00791</sup> mutant ovaries.</p></div>", "links"=>[], "tags"=>["loqs", "steady-state"], "article_id"=>632226, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g002", "stats"=>{"downloads"=>3, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loss_of_Loqs_Function_Increases_the_Steady_State_Concentration_of_Pre_miRNA_/632226", "title"=>"Loss of Loqs Function Increases the Steady-State Concentration of Pre-miRNA", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:03:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/963029"], "description"=>"<p>Testes were stained for DNA (red) and Stellate protein (green). Defects in RNA silencing often lead to accumulation of Stellate protein crystals in testes. For example, the testes from the strong allele <i>armi<sup>72.1</sup>,</i> but not wild-type Oregon R testes, show Stellate protein staining. Testes from <i>loqs</i><sup>f00791</sup> males show strong accumulation of Stellate protein, consistent with their significantly impaired fertility.</p>", "links"=>[], "tags"=>["dsrna-generator", "requires"], "article_id"=>632977, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g009", "stats"=>{"downloads"=>2, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Silencing_of_Stellate_by_the_dsRNA_Generator_Su_Ste_Requires_loqs_/632977", "title"=>"Silencing of <i>Stellate</i> by the dsRNA-Generator <i>Su(Ste)</i> Requires <i>loqs</i>", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:08:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/962938"], "description"=>"<div><p>(A) The red eye color of wild-type flies (left) changes to orange (center) and white (right) in response to one or two copies, respectively, of a <i>white</i> IR transgene, which silences the endogenous <i>white</i> gene.</p>\n <p>(B) Homozygous mutant <i>r2d2</i> flies fail to silence <i>white,</i> even in the presence of two copies of the <i>white</i>-IR transgene; heterozygous <i>r2d2</i>/CyO flies repress <i>white</i> expression.</p>\n <p>(C) In flies homozygous for <i>loqs</i><sup>f00791</sup>, silencing of <i>white</i> by the <i>white</i>-IR is less efficient; two copies of the <i>white</i>-IR do not produce completely white eyes, whereas they do in heterozygous <i>loqs</i><sup>f00791</sup>/CyO.</p>\n <p>(D) The eye color change in <i>loqs</i><sup>f00791</sup> flies is not caused by the increased <i>white<sup>+</sup></i> gene dose resulting from the mini<i>-white</i> marker in the <i>piggyBac</i> transposon that causes the <i>loqs</i><sup>f00791</sup> mutation. Flies <i>trans</i>-heterozygous for <i>loqs</i><sup>f00791</sup> and a mini-<i>white</i>-marked P-element have more red eye pigment than <i>loqs</i><sup>f00791</sup> homozygous flies, but show more efficient silencing by the <i>white</i>-IR than <i>loqs</i><sup>f00791</sup> homozygous animals.</p>\n <p>(E) The eye pigment of the indicated genotypes was extracted and quantified by green light (480 nm) absorbance, relative to wild-type flies bearing no <i>white</i>-IR transgenes. The graph shows the mean and standard deviation of five independent measurements per genotype.</p></div>", "links"=>[], "tags"=>["ir", "depends"], "article_id"=>632890, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g008", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Silencing_of_white_by_an_IR_Partially_Depends_on_loqs_/632890", "title"=>"Silencing of <i>white</i> by an IR Partially Depends on <i>loqs</i>", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:07:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/962803"], "description"=>"<div><p>(A) A YFP transgene expressed from the Pax6-promoter showed strong fluorescence in the eye and weaker fluorescence in the antennae. Due to the underlying normal red eye pigment, the YFP fluorescence was observed in only those ommatidia that are aligned with the optical axis of the stereomicroscope. In heterozygous <i>loqs</i><sup>f00791</sup>/CyO flies bearing a miR-277-responsive, Pax6-promotor-driven, YFP transgene, YFP fluorescence was visible in the antennae but was repressed in the eye. In contrast, in homozygous mutant <i>loqs</i><sup>f00791</sup> flies, YFP fluorescence was readily detected in the eye. A strong mutation in <i>r2d2</i> did not comparably alter repression of the miR-277-regulated YFP reporter. The exposure time for the unregulated YFP reporter strain was one-fourth that used for the miR-277-responsive YFP strain. The exposure times were identical for the heterozygous and homozygous <i>loqs</i> and <i>r2d2</i> flies.</p>\n <p>(B) Additional images of eyes from <i>loqs</i><sup>f00791</sup> heterozygous and homozygous flies bearing the miR-277-responsive YFP reporter transgene diagrammed in (A).</p>\n <p>(C) Quantification of fluorescence of the miR-277-responsive YFP transgene in eyes heterozygous or homozygous for <i>loqs</i> or <i>r2d2</i>. The maximum pixel intensity was measured for each eye (excluding antennae and other tissues where miR-277 does not appear to function). The graph displays the average (<i>n</i> = 13) maximum pixel intensity ± standard deviation for each homozygous genotype, normalized to the average value for the corresponding heterozygotes. Statistical significance was estimated using a two-sample Student's <i>t</i>-test assuming unequal variance.</p>\n <p>The images in (A) were acquired using a sensitive, GFP long-pass filter set that transmits yellow and red autofluorescence. Images in (B) and for quantitative analysis were acquired using a YFP-specific band-pass filter set that reduced the autofluorescence recorded.</p></div>", "links"=>[], "tags"=>["mirna-responsive", "yfp", "requires"], "article_id"=>632762, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g007", "stats"=>{"downloads"=>3, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Silencing_of_a_miRNA_Responsive_YFP_Reporter_Requires_loqs_but_Not_r2d2_/632762", "title"=>"Silencing of a miRNA-Responsive YFP Reporter Requires <i>loqs</i> but Not <i>r2d2</i>", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:06:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/962155"], "description"=>"<div><p>(A) Each of the three <i>D.</i><i>melanogaster</i> RNase III endonucleases pairs with a different dsRBD protein, which assists in its function in RNA silencing.</p>\n <p>(B) Differential splicing creates three <i>loqs</i> mRNA variants, <i>loqs</i> RA, RB, and RC. <i>loqs</i> RA and RB are reported in FlyBase. The RC splice variant is reported here. Arrows mark the position of the PCR primers used in (D); green lines, start codons; red lines, stop codons. The resulting protein isoforms are diagrammed to the right.</p>\n <p>(C) Use of an alternative splice acceptor site extends the 5′ end of exon 4. The mRNA sequence surrounding the new exon–exon junction is shown, with the <i>loqs</i> RC-specific sequence in bold; the arrow marks the position of the last nucleotide of exon 3 relative to the putative transcription start site. When translated into protein, the exon 4 extension inserts 43 new amino acids (indicated below the mRNA sequence) and shifts the Loqs PC reading frame, truncating the protein.</p>\n <p>(D) RT-PCR analysis of <i>loqs</i> mRNA species in males, female carcasses remaining after ovary dissection, dissected ovaries, and S2 cells. Males express more <i>loqs</i> RA than <i>loqs</i> RB, female somatic tissue expresses both <i>loqs</i> RA and <i>loqs</i> RB, while ovaries express predominantly <i>loqs</i> RB. <i>loqs</i> RC was observed only in S2 cells, together with <i>loqs</i> RA and <i>loqs</i> RB.</p>\n <p>(E) The piggyBac transposon insertion f00791 lies 57 bp upstream of the reported transcription start site for <i>loqs</i>.</p></div>", "links"=>[], "tags"=>["dsrbd"], "article_id"=>632134, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biological Sciences", "Cell Biology", "Genetics"], "users"=>["Klaus Förstemann", "Yukihide Tomari", "Tingting Du", "Vasily V Vagin", "Ahmet M Denli", "Diana P Bratu", "Carla Klattenhoff", "William E Theurkauf", "Phillip D Zamore"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.0030236.g001", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loqs_a_dsRBD_Partner_Protein_for_Drosophila_Dcr_1_/632134", "title"=>"Loqs, a dsRBD Partner Protein for <i>Drosophila</i> Dcr-1", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-22 09:02:24"}

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  • {"unique-ip"=>"12", "full-text"=>"10", "pdf"=>"8", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"7", "year"=>"2017", "month"=>"1"}
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  • {"unique-ip"=>"3", "full-text"=>"4", "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"=>"9", "full-text"=>"9", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"5"}
  • {"unique-ip"=>"5", "full-text"=>"5", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"6"}
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  • {"unique-ip"=>"3", "full-text"=>"3", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
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Relative Metric

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