Amyloid Precursor Protein Translation Is Regulated by a 3’UTR Guanine Quadruplex
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{"title"=>"Amyloid precursor protein translation is regulated by a 3'UTR guanine quadruplex", "type"=>"journal", "authors"=>[{"first_name"=>"Ezekiel", "last_name"=>"Crenshaw", "scopus_author_id"=>"56639599900"}, {"first_name"=>"Brian P.", "last_name"=>"Leung", "scopus_author_id"=>"37108263900"}, {"first_name"=>"Chun Kit", "last_name"=>"Kwok", "scopus_author_id"=>"55167223200"}, {"first_name"=>"Michal", "last_name"=>"Sharoni", "scopus_author_id"=>"8976740500"}, {"first_name"=>"Kalee", "last_name"=>"Olson", "scopus_author_id"=>"37057756500"}, {"first_name"=>"Neeraj P.", "last_name"=>"Sebastian", "scopus_author_id"=>"37108365600"}, {"first_name"=>"Sara", "last_name"=>"Ansaloni", "scopus_author_id"=>"8970856100"}, {"first_name"=>"Reinhard", "last_name"=>"Schweitzer-Stenner", "scopus_author_id"=>"7005557639"}, {"first_name"=>"Michael R.", "last_name"=>"Akins", "scopus_author_id"=>"10043910300"}, {"first_name"=>"Philip C.", "last_name"=>"Bevilacqua", "scopus_author_id"=>"7006364103"}, {"first_name"=>"Aleister J.", "last_name"=>"Saunders", "scopus_author_id"=>"7201876732"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84957603024", "pui"=>"608065214", "doi"=>"10.1371/journal.pone.0143160", "sgr"=>"84957603024", "pmid"=>"26618502"}, "id"=>"8ee6d923-f39a-3d9e-bb64-178286391fce", "abstract"=>"A central event in Alzheimer's disease is the accumulation of amyloid β (Aβ) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aβ generation and Alzheimer's disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3'UTR (untranslated region) at residues 3008-3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.", "link"=>"http://www.mendeley.com/research/amyloid-precursor-protein-translation-regulated-3utr-guanine-quadruplex", "reader_count"=>24, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Student > Doctoral Student"=>4, "Researcher"=>3, "Student > Ph. D. Student"=>7, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Student > Doctoral Student"=>4, "Researcher"=>3, "Student > Ph. D. Student"=>7, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>3}, "reader_count_by_subject_area"=>{"Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>4, "Medicine and Dentistry"=>1, "Neuroscience"=>2, "Physics and Astronomy"=>1, "Chemistry"=>2, "Psychology"=>2, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Neuroscience"=>{"Neuroscience"=>2}, "Chemistry"=>{"Chemistry"=>2}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Psychology"=>{"Psychology"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>4}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>4}}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2594207"], "description"=>"<p>(A) Western blot analysis of cells transfected with reporter constructs containing APP <sub>695</sub> coding sequence with wild type (G-quad WT) or mutant (G-quad Mut) sequence. Mock Transfection was used to confirm that these constructs were over-expressed in HeLa cells. Antibody C1/6.1 recognizes both full-length APP<sub>695</sub> and CTF. β-Actin was used as a loading control. (B-C) Quantification of Western blots as in 4A with (B) APP levels normalized to β-Actin and (C) Endogenous CTF intensity values were subtracted from total CTF intensity values to obtain exogenous CTF values which were then normalized to β-Actin values. (D) Aβ ELISA quantification of total Aβ levels from conditioned medium of cells transfected with APP constructs containing the wild type or mutant 3’UTR G-quadruplex.</p>", "links"=>[], "tags"=>["amyloid precursor protein", "Amyloid Precursor Protein Translation", "APP protein expression", "APP expression results", "nm", "pcr", "APP expression", "modulating APP levels", "equivalent mRNA levels", "Western Blot analysis", "utr"], "article_id"=>1614088, "categories"=>["Uncategorised"], "users"=>["Ezekiel Crenshaw", "Brian P. Leung", "Chun Kit Kwok", "Michal Sharoni", "Kalee Olson", "Neeraj P. Sebastian", "Sara Ansaloni", "Reinhard Schweitzer-Stenner", "Michael R. Akins", "Philip C. Bevilacqua", "Aleister J. Saunders"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0143160.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_G_quadruplex_regulation_of_APP_gene_expression_/1614088", "title"=>"G-quadruplex regulation of APP gene expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-11-30 02:57:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/2594209"], "description"=>"<p>(A) Western blot analysis of cells transfected with reporter constructs containing APP <sub>695</sub> coding sequence containing a C-terminal myc tag with either wild type (WT G-quad) or mutant (Mut G-quad) G-quadruplex sequence. Mock transfection was used to confirm that these constructs were over-expressed. Antibody 9B11 (anti-Myc) was used to detect APP-Myc. (B) Schematic representation of methods taken to identify newly synthesized APP (See <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143160#sec002\" target=\"_blank\">methods</a>). (C) Western blot analysis for immunoprecipitation demonstrating the successful pull down of Myc-tagged APP constructs (IP: using 9B11 anti myc mAb, IB: C1/6.1 anti APP). (D) Western blot analysis of total APP (top panel, IB: C1/6.1 anti APP) and newly synthesized APP (bottom panel, IB: Streptavidin). Statistical analysis was performed by normalizing the newly synthesized APP/Total APP (Streptavidin/C1/6.1) using a ratio paired t-test using Prism 6.0g for Mac.</p>", "links"=>[], "tags"=>["amyloid precursor protein", "Amyloid Precursor Protein Translation", "APP protein expression", "APP expression results", "nm", "pcr", "APP expression", "modulating APP levels", "equivalent mRNA levels", "Western Blot analysis", "utr"], "article_id"=>1614090, "categories"=>["Uncategorised"], "users"=>["Ezekiel Crenshaw", "Brian P. Leung", "Chun Kit Kwok", "Michal Sharoni", "Kalee Olson", "Neeraj P. Sebastian", "Sara Ansaloni", "Reinhard Schweitzer-Stenner", "Michael R. Akins", "Philip C. Bevilacqua", "Aleister J. Saunders"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0143160.g005", "stats"=>{"downloads"=>3, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_G_quadruplex_regulation_of_APP_translation_/1614090", "title"=>"G-quadruplex regulation of APP translation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-11-30 02:57:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/2594204"], "description"=>"<p>(A) mRNA levels as assessed by qPCR and represented ΔΔC<sub>T</sub> values represents the normalization of the ΔC<sub>T</sub> of firefly luciferase to the ΔC<sub>T</sub> renilla luciferase then normalized to the wild type. (B) Quantification of Dual Luciferase Assay comparing the wild type G-quadruplex sequence to the mutant G-quadruplex sequence (G-quad Mut). Empty Firefly Luciferase plasmid was used as a control which does not contain the 3’UTR of APP. Firefly Luciferase values were normalized to Renilla Luciferase.</p>", "links"=>[], "tags"=>["amyloid precursor protein", "Amyloid Precursor Protein Translation", "APP protein expression", "APP expression results", "nm", "pcr", "APP expression", "modulating APP levels", "equivalent mRNA levels", "Western Blot analysis", "utr"], "article_id"=>1614084, "categories"=>["Uncategorised"], "users"=>["Ezekiel Crenshaw", "Brian P. Leung", "Chun Kit Kwok", "Michal Sharoni", "Kalee Olson", "Neeraj P. Sebastian", "Sara Ansaloni", "Reinhard Schweitzer-Stenner", "Michael R. Akins", "Philip C. Bevilacqua", "Aleister J. Saunders"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0143160.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_G_quadruplex_regulation_of_Luciferase_gene_expression_/1614084", "title"=>"G-quadruplex regulation of Luciferase gene expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-11-30 02:57:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/2594196"], "description"=>"<p>(A) Schematic of the APP mRNA that contains the putative G-quadruplex sequence in the coding region at position 957 (as identified by Westmark <i>et al</i>. 2007) and the G-quadruplex sequence in the 3’UTR (discussed in this paper) at position 3008. (B) G-quadruplex consensus sequence and comparison of the G-quadruplex sequence in the 3’UTR of human APP with the 3’UTR APP for other species.</p>", "links"=>[], "tags"=>["amyloid precursor protein", "Amyloid Precursor Protein Translation", "APP protein expression", "APP expression results", "nm", "pcr", "APP expression", "modulating APP levels", "equivalent mRNA levels", "Western Blot analysis", "utr"], "article_id"=>1614077, "categories"=>["Uncategorised"], "users"=>["Ezekiel Crenshaw", "Brian P. Leung", "Chun Kit Kwok", "Michal Sharoni", "Kalee Olson", "Neeraj P. Sebastian", "Sara Ansaloni", "Reinhard Schweitzer-Stenner", "Michael R. Akins", "Philip C. Bevilacqua", "Aleister J. Saunders"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0143160.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_APP_contains_a_putative_G_quadruplex_/1614077", "title"=>"APP contains a putative G-quadruplex.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-11-30 02:57:11"}
  • {"files"=>["https://ndownloader.figshare.com/files/2594197"], "description"=>"<p>(A) CD spectra collected as a function of K<sup>+</sup> ion concentration. K<sup>+</sup>-mediated G-quadruplex folding is performed at 2.5 μM RNA under 10 mM lithium cacodylate (LiCac) (pH 7.0), with K<sup>+</sup> ion concentration ranged from 0 to 1 M. The positive peak at ~260 nm and negative peak at ~240 nm are CD signatures for parallel topology of G-quadruplex. (B) CD signal (ellipticity monitored at 262 nm) as a function of K<sup>+</sup> ion concentration from panel A shows clear three-state transitions in G-quadruplex folding. The fitting was performed using <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143160#pone.0143160.e001\" target=\"_blank\">Eq 1</a> (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143160#sec002\" target=\"_blank\">Material and Methods</a>). At physiological K<sup>+</sup> ion concentration (~150 mM), the G-quadruplex is fully folded. The K<sup>+</sup><sub>1/2</sub> and Hill coefficients (n) are provided in the plot. (C) CD titration and comparison of APP 3’UTR wild-type and mutant G-quadruplex sequence. 2.5 μM RNAs were used under 10 mM LiCac (pH 7.0) and physiological 150 mM or 0 mM K<sup>+</sup> ion concentration The GGGG to AAAA substitution in the mutant disfavor G-quadruplex formation as evident by the reduction in CD characteristic signals for G-quadruplex (compare blue and red), and yield similar CD signal to the wild type sequence at 0 mM K<sup>+</sup> ion concentration (green).</p>", "links"=>[], "tags"=>["amyloid precursor protein", "Amyloid Precursor Protein Translation", "APP protein expression", "APP expression results", "nm", "pcr", "APP expression", "modulating APP levels", "equivalent mRNA levels", "Western Blot analysis", "utr"], "article_id"=>1614078, "categories"=>["Uncategorised"], "users"=>["Ezekiel Crenshaw", "Brian P. Leung", "Chun Kit Kwok", "Michal Sharoni", "Kalee Olson", "Neeraj P. Sebastian", "Sara Ansaloni", "Reinhard Schweitzer-Stenner", "Michael R. Akins", "Philip C. Bevilacqua", "Aleister J. Saunders"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0143160.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CD_potassium_ion_titration_of_the_APP_3_UTR_G_quadruplex_shows_that_it_forms_in_vitro_and_is_in_parallel_topology_with_3_state_folding_/1614078", "title"=>"CD potassium ion titration of the <i>APP</i> 3’UTR G-quadruplex shows that it forms <i>in vitro</i> and is in parallel topology with 3-state folding.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-11-30 02:57:11"}

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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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