Structural investig ation of pathog enic RFC1 AA GGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulat edgene expression in CANVAS

Abstract

An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 ( RFC1 ) gene is the genetic cause of cerebellar ataxia, neuropath y, and v estibular arefle xia syndrome (CANVAS), and it also links to se v eral other neurodegenerativ e diseases including the Parkinson's disease. Ho w e v er, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) str uct ure of a bimolecular parallel G4 formed by d(AAGGG) 2 AA and reveal how AAGGG repeats fold into a higher-order str uct ure composed of three G-tetrad la y ers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 str uct ures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented str uct ural basis f or understanding the pathogenic mechanism of AAGGG repeat e xpansion associated with CANVA S . In addition, the high-resolution str uct ures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.