Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

Abstract

CGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

G-quadruplex landscape and its regulation revealed by a new antibody capture method

“…This G4-forming property of DG was lost when it was paired with its reverse complementary oligonucleotide in a Watson-Crick pairing (labeled WC in Fig S11). The hypochromic thermal melting curve (at 260 nm) observed for DG was independent of the KCl concentration, indicating the formation of G4s 49,50 (Fig S11B). Electrophoretic mobility shift assays further indicated that the hybrid G4 conformations preferred by DG were intramolecular (Lanes 2-6 in Fig S11C) as it moved faster than the d(T) 27 oligonucleotide (Lane 1 in Fig S11C).…”

“…These signature peaks were absent in a control WC sequence (Fig S4B), indicating that the hybrid quadruplex formation was specific to Scheme-DG and not observed in the Scheme-WC. The hypochromic thermal melting curve observed for the Scheme-DG was independent of the KCl concentration indicating higher-order G4s formations 45,46 (Fig S4B). Electrophoretic mobility shift assays further indicated that the hybrid G4s conformations preferred by the Scheme-DG were intramolecular (Lanes 2-6 in Fig S4C) as it moved faster than the d(T) 27 (Lane 1 in Fig S4C).…”

“…These signature peaks were absent in a control WC sequence (Fig S4B ), indicating that the hybrid quadruplex formation was specific to Scheme-DG and not observed in the Scheme-WC. The hypochromic thermal melting curve observed for the Scheme-DG was independent of the KCl concentration indicating higher-order G4s formations 45,46 The control DNA could be used in two different ways: (i) added to the sonicated lysate to represent the in vitro false-positive quadruplexes formed without any constraints of the nuclear environment, or (ii) transfected into the cells so that the quadruplexes formation on the same sequence would be subjected to constraints of the nuclear environment (Fig 2A). The differential capture of the control DNA in the two samples would reveal the extent of constraints of the nuclear environment on G4s formation on sequences with canonical G4s forming signatures.…”

Destabilizing Effect of Organo Ru(II) Salts on the Intermolecular Parallel CGG Repeat DNA Quadruplex Associated with Neurodegenerative/Neuromuscular Diseases

“…Solvents were dried by standard methods and distilled before use. The 1 H and 13 C NMR spectra were recorded on Bruker Ultra shield 400 and Bruker AscEnd 600 spectrometers at 25 °C. Chemical shifts, given relative to TMS, were referenced to the solvent resonances as internal standards.…”

Non-canonical DNA/RNA structures associated with the pathogenesis of Fragile X-associated tremor/ataxia syndrome and Fragile X syndrome

“…There is conflicting evidence regarding the secondary structural preference of DNA and RNA strands. CGG stem-loop/hairpins are relatively stable and easily formed in vitro and in vivo using Watson-Crick G:C and Hoogsteen G:G base pairs ( Figure 2A ) ( Chen et al, 1995 ; Mitas et al, 1995 ; Nadel et al, 1995 ; Usdin and Woodford, 1995 ; Yu et al, 1997 ; Handa et al, 2003 ; Sobczak et al, 2003 ; Zumwalt et al, 2007 ; Ciesiolka et al, 2017 ; Ajjugal et al, 2021 ; Poggi and Richard, 2021 ). However, in the presence of physiological K+ concentrations, stable G-quadruplex (G4) and intercalated-motif (i-motif) structures are formed from CGG and CCG repeat strands, respectively ( Figure 2B ) ( Kettani et al, 1995 ; Fojtík and Vorlícková, 2001 ; Weisman-Shomer et al, 2002 ; Weisman-Shomer et al, 2003 ; Khateb et al, 2007 ; Renčiuk et al, 2011 ; Krzyzosiak et al, 2012 ; Loomis et al, 2014 ; Malgowska et al, 2014 ; Yang and Rodgers, 2014 ; Chen et al, 2018 ; Asamitsu et al, 2021 ).…”