Abstract
G-quadruplexes are naturally-occurring structures found in RNAs and DNAs. Regular RNA G-quadruplexes are highly stable due to stacked planar arrangements connected by short loops. However, reports of irregular quadruplex structures are increasing and recent genome-wide studies suggest that they influence gene expression. We have investigated a grouping of G2-motifs in the UTRs of eight genes involved in polyamine biosynthesis, and concluded that several likely form novel metastable RNA G-quadruplexes. We performed a comprehensive biophysical characterization of their properties, comparing them to a reference G-quadruplex. Using cellular assays, together with polyamine-depleting and quadruplex-stabilizing ligands, we discovered how some of these motifs regulate and sense polyamine levels, creating feedback loops during polyamine biosynthesis. Using high-resolution 1H-NMR spectroscopy, we demonstrated that a long-looped quadruplex in the AZIN1 mRNA co-exists in salt-dependent equilibria with a hairpin structure. This study expands the repertoire of regulatory G-quadruplexes and demonstrates how they act in unison to control metabolite homeostasis.
Highlights
Polyamines (PAs) are small poly-cationic molecules present at millimolar concentrations in cells (Lightfoot and Hall, 2014)
PAdirected feedback loops operate at RNA and protein levels, whereby low levels of PAs are corrected with increased expression of PA synthesis enzymes and decreased activities of negative regulators (Miller-Fleming et al, 2015; Ivanov et al, 2010; Perez-Leal and Merali, 2012)
We searched for putative quadruplex structures (PQS’s) in the UTRs of PA synthesis proteins (PSPs) (Figure 1a) using the algorithm QGRS Mapper (Kikin et al, 2006), which predicts the ability of a sequence containing G-repeats to fold into distinct quadruplexes and assigns them a stability score (G-score) based on published biophysical data
Summary
Polyamines (PAs) are small poly-cationic molecules present at millimolar concentrations in cells (Lightfoot and Hall, 2014). PAdirected feedback loops operate at RNA and protein levels, whereby low levels of PAs are corrected with increased expression of PA synthesis enzymes and decreased activities of negative regulators (Miller-Fleming et al, 2015; Ivanov et al, 2010; Perez-Leal and Merali, 2012). ODC1 is the limiting factor in PA synthesis (Miller-Fleming et al, 2015): it has a highly structured 5’UTR and its translation is strongly affected by PA levels. It is negatively regulated by binding to OAZ proteins, which themselves are regulated by binding AZIN1. PAs bind to nascent OAZ polypeptides, activating a frameshift needed for production of full-length OAZ (Ivanov et al, 2010; Kurian et al, 2011)
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