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Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

Crenshaw E, Leung BP, Kwok CK, Sharoni M, Olson K, Sebastian NP, Ansaloni S, Schweitzer-Stenner R, Akins MR, Bevilacqua PC, Saunders AJ - PLoS ONE (2015)

Bottom Line: 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.To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation.Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Drexel University, Philadelphia, PA, United States of America.

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.

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G-quadruplex regulation of APP translation.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 methods). (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.
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pone.0143160.g005: G-quadruplex regulation of APP translation.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 methods). (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.

Mentions: Loss of the G-quadruplex leads to an increase in APP protein levels (Fig 4) without affecting the APP transcript levels (Fig 3A), suggesting a role for this structure in translational control. To test this prediction, we labeled newly synthesized proteins during a discrete window and asked whether more APP protein was produced from the construct in which the G-quadruplex was mutated. For these experiments, we used HeLa cells that expressed myc-tagged APP constructs followed by either the wild type or G-quadruplex-mutated 3’ UTR (Fig 5A). These cells were metabolically labeled for four hours with L-azidohomoalanine (AHA), a methionine analog that is incorporated into newly synthesized proteins during translation and can be subsequently detected using click chemistry-based approaches. In this approach a desthiobiotin molecule containing an alkyne group forms a covalent bond to the AHA molecule incorporated into newly synthesized proteins (Fig 5B) [52]. Specifically, we immunoprecipitated the exogenous APP using antibodies that recognize myc, biotinylated the AHA-containing APP, and used immunoblotting to determine the extent to which this immunoprecipitated APP had been synthesized during the labeling window (Fig 5C). Using this approach, we determined that the mutant construct resulted in an increase in newly synthesized APP of 19.4% ± 5.3% (mean ± SEM; ratio paired t-test p = 0.0052; n = 6) during the labeling window (Fig 5D). Taken together with the previous findings, these results demonstrate that the 3’UTR G-quadruplex modulates APP protein expression by negatively regulating translation.


Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

Crenshaw E, Leung BP, Kwok CK, Sharoni M, Olson K, Sebastian NP, Ansaloni S, Schweitzer-Stenner R, Akins MR, Bevilacqua PC, Saunders AJ - PLoS ONE (2015)

G-quadruplex regulation of APP translation.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 methods). (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.
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Related In: Results  -  Collection

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pone.0143160.g005: G-quadruplex regulation of APP translation.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 methods). (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.
Mentions: Loss of the G-quadruplex leads to an increase in APP protein levels (Fig 4) without affecting the APP transcript levels (Fig 3A), suggesting a role for this structure in translational control. To test this prediction, we labeled newly synthesized proteins during a discrete window and asked whether more APP protein was produced from the construct in which the G-quadruplex was mutated. For these experiments, we used HeLa cells that expressed myc-tagged APP constructs followed by either the wild type or G-quadruplex-mutated 3’ UTR (Fig 5A). These cells were metabolically labeled for four hours with L-azidohomoalanine (AHA), a methionine analog that is incorporated into newly synthesized proteins during translation and can be subsequently detected using click chemistry-based approaches. In this approach a desthiobiotin molecule containing an alkyne group forms a covalent bond to the AHA molecule incorporated into newly synthesized proteins (Fig 5B) [52]. Specifically, we immunoprecipitated the exogenous APP using antibodies that recognize myc, biotinylated the AHA-containing APP, and used immunoblotting to determine the extent to which this immunoprecipitated APP had been synthesized during the labeling window (Fig 5C). Using this approach, we determined that the mutant construct resulted in an increase in newly synthesized APP of 19.4% ± 5.3% (mean ± SEM; ratio paired t-test p = 0.0052; n = 6) during the labeling window (Fig 5D). Taken together with the previous findings, these results demonstrate that the 3’UTR G-quadruplex modulates APP protein expression by negatively regulating translation.

Bottom Line: 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.To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation.Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Drexel University, Philadelphia, PA, United States of America.

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.

Show MeSH
Related in: MedlinePlus