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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 gene expression.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 APP695 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.
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pone.0143160.g004: G-quadruplex regulation of APP gene expression.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 APP695 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.

Mentions: We next sought to establish a system in which we could measure the effects of this G-quadruplex on APP expression. For these experiments, we used a plasmid encoding the 695 amino acid isoform of human APP (APP695) followed by either the wild-type- or G-quadruplex-mutated human APP 3’UTR. These constructs were transiently transfected separately into HeLa cells, which do not express the 695 amino acid isoform of APP [50, 51]. Cell lysates were collected 24 hours post-transfection and subjected to Western blot analysis. Consistent with previous findings [50, 51], we could not detect APP695 in untransfected cells (Fig 4A). In contrast, transfected cells expressed readily detectable APP695 (Fig 4A). Therefore we can specifically detect exogenous full-length APP695 using Western blot analysis.


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 gene expression.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 APP695 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664259&req=5

pone.0143160.g004: G-quadruplex regulation of APP gene expression.(A) Western blot analysis of cells transfected with reporter constructs containing APP 695 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 APP695 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.
Mentions: We next sought to establish a system in which we could measure the effects of this G-quadruplex on APP expression. For these experiments, we used a plasmid encoding the 695 amino acid isoform of human APP (APP695) followed by either the wild-type- or G-quadruplex-mutated human APP 3’UTR. These constructs were transiently transfected separately into HeLa cells, which do not express the 695 amino acid isoform of APP [50, 51]. Cell lysates were collected 24 hours post-transfection and subjected to Western blot analysis. Consistent with previous findings [50, 51], we could not detect APP695 in untransfected cells (Fig 4A). In contrast, transfected cells expressed readily detectable APP695 (Fig 4A). Therefore we can specifically detect exogenous full-length APP695 using Western blot analysis.

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