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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 Luciferase gene expression.(A) mRNA levels as assessed by qPCR and represented ΔΔCT values represents the normalization of the ΔCT of firefly luciferase to the ΔCT 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.
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pone.0143160.g003: G-quadruplex regulation of Luciferase gene expression.(A) mRNA levels as assessed by qPCR and represented ΔΔCT values represents the normalization of the ΔCT of firefly luciferase to the ΔCT 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.

Mentions: We next investigated the functional significance of this G-quadruplex. As these structures can play roles in transcription, RNA stability and translation[28], we wanted to investigate what role the APP 3’UTR G-quadruplex has on gene expression. We began these studies by investigating whether the G-quadruplex regulated RNA levels, which would suggest a role in either transcription and/or RNA stability. To perform these studies, we utilized a luciferase reporter construct in which the human APP 3’UTR was inserted after the stop codon of the firefly luciferase gene [49]. To gain insight into the role of the G-quadruplex in regulating expression we created a parallel luciferase construct in which we disrupted the 3’UTR G-quadruplex structure by changing the fourth set of guanine repeats to adenines thereby disrupting tetrad formation [40, 48]; this mutant is analogous to the mutant oligonucleotide sequence used in the CD studies above. To test whether the G-quadruplex affected the expression levels of these transcripts, we transfected HEK293 cells with either construct using identical transfection conditions Using qPCR to measure luciferase mRNA levels in these cell populations, we could detect no differences between luciferase mRNA having either the wild type or mutant G-quadruplex (Fig 3A). The APP 3’UTR G-quadruplex therefore does not play any substantial role in regulating the expression at the level of transcription or RNA stability.


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 Luciferase gene expression.(A) mRNA levels as assessed by qPCR and represented ΔΔCT values represents the normalization of the ΔCT of firefly luciferase to the ΔCT 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664259&req=5

pone.0143160.g003: G-quadruplex regulation of Luciferase gene expression.(A) mRNA levels as assessed by qPCR and represented ΔΔCT values represents the normalization of the ΔCT of firefly luciferase to the ΔCT 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.
Mentions: We next investigated the functional significance of this G-quadruplex. As these structures can play roles in transcription, RNA stability and translation[28], we wanted to investigate what role the APP 3’UTR G-quadruplex has on gene expression. We began these studies by investigating whether the G-quadruplex regulated RNA levels, which would suggest a role in either transcription and/or RNA stability. To perform these studies, we utilized a luciferase reporter construct in which the human APP 3’UTR was inserted after the stop codon of the firefly luciferase gene [49]. To gain insight into the role of the G-quadruplex in regulating expression we created a parallel luciferase construct in which we disrupted the 3’UTR G-quadruplex structure by changing the fourth set of guanine repeats to adenines thereby disrupting tetrad formation [40, 48]; this mutant is analogous to the mutant oligonucleotide sequence used in the CD studies above. To test whether the G-quadruplex affected the expression levels of these transcripts, we transfected HEK293 cells with either construct using identical transfection conditions Using qPCR to measure luciferase mRNA levels in these cell populations, we could detect no differences between luciferase mRNA having either the wild type or mutant G-quadruplex (Fig 3A). The APP 3’UTR G-quadruplex therefore does not play any substantial role in regulating the expression at the level of transcription or RNA stability.

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