RESUMO
The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Quadruplex G , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Camundongos , Ligação Proteica , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/químicaRESUMO
A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNAâ¢DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.
Assuntos
Expansão das Repetições de DNA/genética , Fases de Leitura Aberta/genética , Esclerose Lateral Amiotrófica/genética , Linfócitos B , Sequência de Bases , Nucléolo Celular/genética , Nucléolo Celular/patologia , DNA/genética , DNA/metabolismo , Demência Frontotemporal/genética , Quadruplex G , Células HEK293 , Humanos , Modelos Moleculares , Neurônios , Fosfoproteínas/metabolismo , RNA/biossíntese , RNA/química , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Estresse Fisiológico , Transcrição Gênica/genética , NucleolinaRESUMO
The C9orf72 hexanucleotide repeat expansion (HRE) is the most frequent genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we describe the pathogenic cascades that are initiated by the C9orf72 HRE DNA. The HRE DNA binds to its protein partner DAXX and promotes its liquid-liquid phase separation, which is capable of reorganizing genomic structures. An HRE-dependent nuclear accumulation of DAXX drives chromatin remodeling and epigenetic changes such as histone hypermethylation and hypoacetylation in patient cells. While regulating global gene expression, DAXX plays a key role in the suppression of basal and stress-inducible expression of C9orf72 via chromatin remodeling and epigenetic modifications of the promoter of the major C9orf72 transcript. Downregulation of DAXX or rebalancing the epigenetic modifications mitigates the stress-induced sensitivity of C9orf72-patient-derived motor neurons. These studies reveal a C9orf72 HRE DNA-dependent regulatory mechanism for both local and genomic architectural changes in the relevant diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , DNA/genética , DNA/metabolismo , Expansão das Repetições de DNA/genética , Epigênese Genética , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Histonas/metabolismoRESUMO
RNA plays an active role in structural polymorphism of the genome through the formation of stable RNAâ¢DNA hybrids (R-loops). R-loops can modulate normal physiological processes and are also associated with pathological conditions, such as those related to nucleotide repeat expansions. A guanine-rich hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been linked to a spectrum of neurological conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we discuss the possible roles, both locally and genome-wide, of R-loops that may arise from the C9orf72 hexanucleotide repeat. R-loops have the potential to influence the pathological processes identified in many repeat expansion diseases, such as repeat instability, transcriptional dysregulation, epigenetic modification, and antisense-mediated gene regulation. We propose that, given the wide-ranging consequences of R-loops in the cell, these structures could underlie multiple pathological processes in C9orf72-linked neurodegeneration.