Control of translation initiation and neuronal subcellular localisation of mRNAs by G-quadruplex structures

Abstract

The translation of mRNA is a key regulatory step in control of gene expression. The primary sequence of an mRNA determines much of the regulation of translation, including the location of translation initiation. The efficiency of translation initiation is determined by the initiation codon and its context. Translation initiation occurs at the canonical initiation codon, AUG, or a less efficient near canonical non-AUG alternative initiation codon (AIC). The secondary structure of an mRNA impacts its translation, principally by inhibiting binding and/or migration of ribosomal subunits. Hairpins form by hydrogen bonding between Watson-Crick complementary base-pairs. Guanine-rich nucleotide sequences form planar tetrads via hydrogen bonds and stack as stable G-quadruplex structures. Much has been reported on the impact of hairpins in regulating translation and G-quadruplex structures are emerging as an important factor in the regulation of translation of some mRNAs.

Two-pore potassium leak (K2P) channels set and maintain the resting membrane potential of cells, so precise regulation of K2P protein expression in cells is critical to cell behaviour and survival. The 5’ untranslated regions (5’ UTR) of K2P channels are poorly annotated in databases. This thesis details investigations of 5’ UTR primary sequence and secondary structure effects on the expression of K2P leak channels. We characterised an extension to the annotated 5’ UTR sequence of Task3 by 5’RACE. The extension is characterised by a 5’ terminal (GGN) repeat which we show forms a G-quadruplex structure and inhibits translation of Task 3. G-quadruplex formation was measured by circular dichroism and increased fluorescence of Mesoporphyrin IX dihydrochloride. RT-PCR of Task3-FLAG mRNA relative to ?-actin mRNA in polysome fractions of transfected HeLa cells suggests the inhibition of Task3 protein synthesis results from an inhibition of translation initiation due to the 5’-terminal G-quadruplex. Results suggest the inhibition of Task3 protein synthesis can be modulated by RNA-binding proteins. hnRNPA2 is shown to relieve translational inhibition, and the DEAH/RHA RNA helicases, DHX29, DHX30 and DHX36 differentially regulate translation from Task3 mRNA dependent on the presence of the 5’ terminal G-quadruplex. We also investigate the use of small ligands TMPyP4 and Hippuristanol in modulating expression of Task3 in a G-quadruplex-dependent manner.

Task3 is expressed in neuronal cells and is predicted to be able to exert control over local membrane potential. The local translation of mRNAs has been demonstrated in a variety of cells. In neurons, local translation of some mRNAs at synapses have been shown in plastic changes. G-quadruplex structures have been shown to affect mRNA subcellular targeting and direct protein synthesis at the synapse. We describe detection of Task3-GFP mRNA by Fluorescence In Situ Hybridisation (FISH) in transfected primary cortical neurons. It was found for the first time that the 5’-terminal G-quadruplex in Task3 mRNA appears to mediate localisation of Task3-GFP mRNA to discrete neurite particles. The roles of G-quadruplex-interacting RNA binding proteins (RBPs), hnRNP A2 and Pur[alpha], in the trafficking of Task3 mRNA was investigated, however, the mechanism of neurite delivery of Task3 mRNA requires further research. Dysregulation of this mechanism would cause perturbations to individual synapse excitability and therefore contribute to neuronal behaviour.

FXR2 is a paralog of the neuronal translation regulator RBP, Fragile X Mental Retardation Protein (FMRP). Ribosome profiling of mouse embryonic stem cells identified Fxr2 to be subject to alternative upstream translation initiation. Results here demonstrate the production of different N-terminal length isoforms of Fxr2 from alternative translation initiation, primarily from GUG codon, -219 from the annotated AUG initiation codon. Investigation of the 5’ UTR of Fxr2 revealed a high concentration of guanine residues. We show evidence supporting G-quadruplex structures within its 5’UTR. This is the first report of potential G-quadruplex mediated control of AIC usage in an mRNA.

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ORCID for Mark Coldwell: orcid.org/0000-0002-6243-3886

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