Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease which results in progressive degeneration of motor neurons and for which there is no cure. Patients diagnosed with ALS have been shown to have a decreased abundance of a nucleic acid binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNP K), as well as a microRNA (miR), miR-1249-3p. Previous work reveals that miR-1249-3p regulates the translation of hnRNP K mRNA which expresses the HNRNP K protein by binding to its 3’ untranslated region. HnRNP K pays a role in the homeostasis of neurons, so a decrease in this protein may be a causative agent of ALS. We have previously identified that a G quadruplex (GQ) structure forms in this 3’-untranslated region of hnRNP K mRNA, which also encompasses the miR-1249-3p binding site. In this study we postulate that this GQ structure may alter the binding affinity of the miR-1249-3p and give reason for the dysregulation of hnRNP K protein in ALS patients. Further, fragile X mental retardation protein (FMRP) has been shown to bind GQ structures in RNA and interact with the miR pathway, suggesting it may have cooperative effects on mediating miR-1249-3p and hnRNP K RNA binding. To test this hypothesis, we used electrophoretic mobility shift assays, <sup>1</sup>H NMR spectroscopy ,and fluorescence spectroscopy studies to provide biophysical information about the stability of the hnRNP K mRNA GQ in different conditions as well as this structure’s ability to modulate the binding of the miR-1249-3p. These experiments demonstrated that the GQ was unstable in the absence of KCl, enabling the binding of miR-1249-3p, and further demonstrated that this interaction may explain the downregulation of hnRNP K and miR-1249-3p in ALS.

The interactions between HNRNP K mRNA and miR-1249-3P are mediated by a noncanonical G quadruplex structure

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