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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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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.(A) CD spectra collected as a function of K+ ion concentration. K+-mediated G-quadruplex folding is performed at 2.5 μM RNA under 10 mM lithium cacodylate (LiCac) (pH 7.0), with K+ 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+ ion concentration from panel A shows clear three-state transitions in G-quadruplex folding. The fitting was performed using Eq 1 (see Material and Methods). At physiological K+ ion concentration (~150 mM), the G-quadruplex is fully folded. The K+1/2 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+ 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+ ion concentration (green).
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pone.0143160.g002: 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.(A) CD spectra collected as a function of K+ ion concentration. K+-mediated G-quadruplex folding is performed at 2.5 μM RNA under 10 mM lithium cacodylate (LiCac) (pH 7.0), with K+ 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+ ion concentration from panel A shows clear three-state transitions in G-quadruplex folding. The fitting was performed using Eq 1 (see Material and Methods). At physiological K+ ion concentration (~150 mM), the G-quadruplex is fully folded. The K+1/2 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+ 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+ ion concentration (green).

Mentions: To validate that the sequence identified by the bioinformatic approach is a bona fide G-quadruplex, we performed a structural characterization of this sequence. Several factors contribute to the folding of an RNA into a G-quadruplex, including the sequence itself (guanine tracts, loop sequence, and loop length) as well as the cellular environment (pH, temperature, and the concentration and identities of monovalent cations) [31, 43–45]. Importantly, potassium (K+) ions preferentially stabilizes G-quadruplex structures in comparison to sodium (Na+) and lithium (Li+) ions [46]. G-quadruplex formation can be monitored through key spectral signatures using circular dichroism (CD) spectroscopy. To test whether the 3’UTR sequence forms a G-quadruplex, we used an RNA oligonucleotide bearing the putative APP 3’UTR G-quadruplex and performed K+ ion titration monitored by CD. The first immediate observation is that CD spectra of this RNA includes a negative peak at 240 nm and a positive peak at 262 nm (Fig 2A), which are the distinctive CD signatures for a parallel G-quadruplex structure [38, 47]. We next investigated the K+ ion dependence of this G-quadruplex structure. Plotting the change in ellipticity versus K+ ion concentration revealed a three-state transition (Fig 2B), with a K+1/2 of ~3 μM and ~18 mM. As the physiological K+ concentration is ~150 mM, this result suggests that the APP 3’UTR G-quadruplex is fully folded in vivo, with a maximum of 3-quartet planes. We then used CD to compare the oligonucleotide representing the wild-type APP 3’UTR G-quadruplex to the spectrum of the oligonucleotide representing a APP 3’UTR G-quadruplex sequence in which the fourth set of G repeats was replaced with adenines (APP 3’UTR G-Quad Mutant) [40, 48]. This was conducted in the presence of 150 mM KCl, which induces G-quadruplex formation in the wild-type sequence. In this mutated oligonucleotide we observed a significant decrease in the 262 nm peak, indicating a decrease in population of the G-quadruplex fold (Fig 2C). Taken together, our CD data support the presence of a parallel G-quadruplex that is stabilized by K+ ions.


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)

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.(A) CD spectra collected as a function of K+ ion concentration. K+-mediated G-quadruplex folding is performed at 2.5 μM RNA under 10 mM lithium cacodylate (LiCac) (pH 7.0), with K+ 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+ ion concentration from panel A shows clear three-state transitions in G-quadruplex folding. The fitting was performed using Eq 1 (see Material and Methods). At physiological K+ ion concentration (~150 mM), the G-quadruplex is fully folded. The K+1/2 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+ 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+ ion concentration (green).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664259&req=5

pone.0143160.g002: 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.(A) CD spectra collected as a function of K+ ion concentration. K+-mediated G-quadruplex folding is performed at 2.5 μM RNA under 10 mM lithium cacodylate (LiCac) (pH 7.0), with K+ 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+ ion concentration from panel A shows clear three-state transitions in G-quadruplex folding. The fitting was performed using Eq 1 (see Material and Methods). At physiological K+ ion concentration (~150 mM), the G-quadruplex is fully folded. The K+1/2 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+ 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+ ion concentration (green).
Mentions: To validate that the sequence identified by the bioinformatic approach is a bona fide G-quadruplex, we performed a structural characterization of this sequence. Several factors contribute to the folding of an RNA into a G-quadruplex, including the sequence itself (guanine tracts, loop sequence, and loop length) as well as the cellular environment (pH, temperature, and the concentration and identities of monovalent cations) [31, 43–45]. Importantly, potassium (K+) ions preferentially stabilizes G-quadruplex structures in comparison to sodium (Na+) and lithium (Li+) ions [46]. G-quadruplex formation can be monitored through key spectral signatures using circular dichroism (CD) spectroscopy. To test whether the 3’UTR sequence forms a G-quadruplex, we used an RNA oligonucleotide bearing the putative APP 3’UTR G-quadruplex and performed K+ ion titration monitored by CD. The first immediate observation is that CD spectra of this RNA includes a negative peak at 240 nm and a positive peak at 262 nm (Fig 2A), which are the distinctive CD signatures for a parallel G-quadruplex structure [38, 47]. We next investigated the K+ ion dependence of this G-quadruplex structure. Plotting the change in ellipticity versus K+ ion concentration revealed a three-state transition (Fig 2B), with a K+1/2 of ~3 μM and ~18 mM. As the physiological K+ concentration is ~150 mM, this result suggests that the APP 3’UTR G-quadruplex is fully folded in vivo, with a maximum of 3-quartet planes. We then used CD to compare the oligonucleotide representing the wild-type APP 3’UTR G-quadruplex to the spectrum of the oligonucleotide representing a APP 3’UTR G-quadruplex sequence in which the fourth set of G repeats was replaced with adenines (APP 3’UTR G-Quad Mutant) [40, 48]. This was conducted in the presence of 150 mM KCl, which induces G-quadruplex formation in the wild-type sequence. In this mutated oligonucleotide we observed a significant decrease in the 262 nm peak, indicating a decrease in population of the G-quadruplex fold (Fig 2C). Taken together, our CD data support the presence of a parallel G-quadruplex that is stabilized by K+ ions.

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