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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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APP contains a putative G-quadruplex.(A) Schematic of the APP mRNA that contains the putative G-quadruplex sequence in the coding region at position 957 (as identified by Westmark et al. 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.
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pone.0143160.g001: APP contains a putative G-quadruplex.(A) Schematic of the APP mRNA that contains the putative G-quadruplex sequence in the coding region at position 957 (as identified by Westmark et al. 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.

Mentions: To investigate the post-transcriptional regulation of APP expression, we asked whether the human APP mRNA contains a G-quadruplex. Using Quadparser [36], we searched the APP mRNA sequence (NM_201414.2) for putative G-quadruplex sequences following the sequence motif, (G≥ 2N1–7) 3G≥ 2 which defines four repeats of at least two guanines (G) interrupted by stretches of one to seven nucleotides of any type (N). This approach predicted two putative G-quadruplexes within APP mRNA (Fig 1A). One such sequence is located within the protein coding region beginning at nucleotide 957, consistent with earlier findings [29]. This potential G-quadruplex is predicted to be relatively weak since it has the potential to form a quadruplex with only two stacks of guanine tetrads and the intervening loops are relatively long with 4 nucleotides each [41]. A second putative G-quadruplex was identified within the 3’UTR beginning at nucleotide 3008. This 3’UTR sequence was recently identified independently by a bioinformatic analysis but was not experimentally investigated [42]. This putative G-quadruplex could form a relatively strong G-quadruplex since it has the potential to form a quadruplex comprised of three guanine tetrads and the intervening loops are only 2 or 1 nucleotides in length [31]. Since the G-quadruplex found in APP 3’UTR is approximately 718 nucleotides from the stop codon, we searched the APP gene of several species for potential G-quadruplexes around the same nucleotide distance from the stop codon and was able to obtain an alignment for the comparison of the genes using GQRS-H Predictor software [37] (Fig 1B). The potential functional importance of this 3’UTR G-quadruplex is highlighted by its conservation in APP genes in various species (Fig 1B). These findings suggest the presence of functional G-quadruplexes in the APP mRNA.


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)

APP contains a putative G-quadruplex.(A) Schematic of the APP mRNA that contains the putative G-quadruplex sequence in the coding region at position 957 (as identified by Westmark et al. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143160.g001: APP contains a putative G-quadruplex.(A) Schematic of the APP mRNA that contains the putative G-quadruplex sequence in the coding region at position 957 (as identified by Westmark et al. 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.
Mentions: To investigate the post-transcriptional regulation of APP expression, we asked whether the human APP mRNA contains a G-quadruplex. Using Quadparser [36], we searched the APP mRNA sequence (NM_201414.2) for putative G-quadruplex sequences following the sequence motif, (G≥ 2N1–7) 3G≥ 2 which defines four repeats of at least two guanines (G) interrupted by stretches of one to seven nucleotides of any type (N). This approach predicted two putative G-quadruplexes within APP mRNA (Fig 1A). One such sequence is located within the protein coding region beginning at nucleotide 957, consistent with earlier findings [29]. This potential G-quadruplex is predicted to be relatively weak since it has the potential to form a quadruplex with only two stacks of guanine tetrads and the intervening loops are relatively long with 4 nucleotides each [41]. A second putative G-quadruplex was identified within the 3’UTR beginning at nucleotide 3008. This 3’UTR sequence was recently identified independently by a bioinformatic analysis but was not experimentally investigated [42]. This putative G-quadruplex could form a relatively strong G-quadruplex since it has the potential to form a quadruplex comprised of three guanine tetrads and the intervening loops are only 2 or 1 nucleotides in length [31]. Since the G-quadruplex found in APP 3’UTR is approximately 718 nucleotides from the stop codon, we searched the APP gene of several species for potential G-quadruplexes around the same nucleotide distance from the stop codon and was able to obtain an alignment for the comparison of the genes using GQRS-H Predictor software [37] (Fig 1B). The potential functional importance of this 3’UTR G-quadruplex is highlighted by its conservation in APP genes in various species (Fig 1B). These findings suggest the presence of functional G-quadruplexes in the APP mRNA.

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