RNA G-Quadruplexes in the model plant species Arabidopsis thaliana: prevalence and possible functional roles.

Tandem stretches of guanines can associate in hydrogen-bonded arrays to form G-quadruplexes, which are stabilized by K(+) ions. Using computational methods, we searched for G-Quadruplex Sequence (GQS) patterns in the model plant species Arabidopsis thaliana. We found ∼ 1200 GQS with a G(3) repeat sequence motif, most of which are located in the intergenic region. Using a Markov modeled genome, we determined that GQS are significantly underrepresented in the genome. Additionally, we found ∼ 43,000 GQS with a G(2) repeat sequence motif; notably, 80% of these were located in genic regions, suggesting that these sequences may fold at the RNA level. Gene Ontology functional analysis revealed that GQS are overrepresented in genes encoding proteins of certain functional categories, including enzyme activity. Conversely, GQS are underrepresented in other categories of genes, notably those for non-coding RNAs such as tRNAs and rRNAs. We also find that genes that are differentially regulated by drought are significantly more likely to contain a GQS. CD-detected K(+) titrations performed on representative RNAs verified formation of quadruplexes at physiological K(+) concentrations. Overall, this study indicates that GQS are present at unique locations in Arabidopsis and that folding of RNA GQS may play important roles in regulating gene expression.

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

A central event in Alzheimer's disease is the accumulation of amyloid ß (Aß) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aß 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 Aß 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 Aß 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.