C9orf72 and triplet repeat disorder RNAs: G-quadruplex formation, binding to PRC2 and implications for disease mechanisms

Abstract

Some neurological disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, Huntington's disease, myotonic dystrophy, and various ataxias, can be caused by expansions of short nucleic acid sequence repeats in specific genes. A possible disease mechanism involves the transcribed repeat RNA binding an RNAbinding protein (RBP), resulting in its sequestration and thus dysfunction. Polycomb repressive complex 2 (PRC2), the histone methyltransferase that deposits the H3K27me3 mark of epigenetically silenced chromatin, binds G-rich RNAs and has especially high affinity for G-quadruplex (G-Q) structures. Here, we find that PRC2 target genes are derepressed and the RNA binding subunit EZH2 largely insoluble in postmortem brain samples from ALS/FTD patients with C9ORF72 (C9) repeat expansions, leading to the hypothesis that the (G 4 C 2 ) n repeat RNA might be sequestering PRC2. Contrary to this expectation, we found that C9 repeat RNAs (n = 6 or 10) bind weakly to purified PRC2, and studies with the G-Q specific BG4 antibody and circular dichroism studies both indicated that these C9 RNAs have little propensity to form G-Qs in vitro. Several GC-rich triplet-repeat expansion RNAs also have low affinity for PRC2 and do not appreciably form G-Qs in vitro. The results are consistent with these sequences forming hairpin structures that outcompete G-Q folding when the repeat length is sufficiently large. We suggest that binding of PRC2 to these GC-rich RNAs is fundamentally weak but may be modulated in vivo by protein factors that affect secondary structure, such as helicases and other RBPs.

CGG repeat RNA G-quadruplexes interact with FMRpolyG to cause neuronal dysfunction in fragile X-related tremor/ataxia syndrome

“…In G4-nonstabilizing Li + conditions, positive and negative CD absorption bands at 262 and 210 nm were detected, which were characteristic of an A-form duplex RNA conformation. However, in G4-stabilizing K + conditions, a negative band at 210 nm was recovered toward the positive side, which is consistent with G4 conformation ( 22 ). In CGG17 RNA prepared with the same concentration as CGG99 RNA, CD measurements indicated lower absorptions at 262 and 210 nm compared with CGG99 RNA under Li + and K + conditions, showing a smaller proportion of rigid secondary structures than those of CGG99 RNA.…”

Functional Roles of Long Non-coding RNAs in Motor Neuron Development and Disease

“…This outcome may indicate that C9ORF72-AS RNA may also contribute to the pathology caused by C9ORF72 hexanucleotide repeat expansion. Whereas C9ORF72-S can form G-quadruplexes [46,55,116] that are known to regulate transcription and gene expression [150], the C-rich C9ORF72-AS repeats may not form similar structures. Instead, the G 2 C 4 expansions in C9ORF72-AS may form a C-rich motif [65] that likely affects genome stability and transcription [1].…”

Exploring Computational and Biophysical Tools to Study the Presence of G-Quadruplex Structures: A Promising Therapeutic Solution for Drug-Resistant Vibrio cholerae

“…Similarly, stabilization of GQ motif causes an unusual −1 ribosomal frameshift in mammalian cells ( Endoh and Sugimoto, 2013 ). G-quadruplex motifs in the C9orf72 are responsible for the blockade of various functional proteins responsible for neurodegenerative diseases and are one of the main causes of Amyotrophic lateral sclerosis and frontotemporal dementia ( Wang et al, 2019 ). In bacterial species, the presence of GQ motifs in the open reading frame have been shown to have an impeding effect on the transcription of the PGQ harboring genes ( Mishra et al, 2019a , b ).…”