CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

The RNA helicase DHX36-G4R1 modulates C9orf72 GGGGCC hexanucleotide repeat-associated translation.

GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.

Exogenous polyserine fibrils change membrane properties of phosphatidylcholine-liposome and red blood cells.

The causative genes for neurodegenerative polyglutamine (polyQ) diseases produce homopolymeric polyglutamine (polyQ), polyserine (polyS), polyalanine (polyA), polycysteine (polyC), and polyleucine (polyL) sequences by repeat-associated non-AUG (RAN) translation. The cytotoxicity of the intracellular polyQ and RAN products has been extensively investigated. However, little is known about the toxicity of the extracellular polyQ and RAN products on the membranes of viable cells. Because polyQ aggregates induce a deflated morphology of a model membrane, we hypothesized that extracellular polyQ and RAN products might affect the membrane properties of viable cells. In this study, we demonstrated that exogenous polyS fibrils but not polyS or polyQ non-fibril aggregates altered the thermal phase transition behavior of a model membrane composed of a phosphatidylcholine bilayer using differential scanning calorimetry. PolyS fibrils induced morphological changes in viable red blood cells (RBCs). However, both polyS and polyQ non-fibril aggregates had no effects on RBCs. These results highlight the possibility that extracellular fibrils generated from RAN products may alter the properties of neuronal cell membranes, which may contribute to changes in the brain pathology.

RNA Dysmetabolism and Repeat-Associated Non-AUG Translation in Frontotemporal Lobar Degeneration/Amyotrophic Lateral Sclerosis due to C9orf72 Hexanucleotide Repeat Expansion.

Neuropathological features of frontotemporal dementia and amyotrophic lateral sclerosis (ALS) due to C9orf72 GGGGCC hexanucleotide repeat expansion include early dipeptide repeats, repeat RNA foci, and subsequent TDP-43 pathologies. Since the discovery of the repeat expansion, extensive studies have elucidated the disease mechanism of how the repeat causes neurodegeneration. In this review, we summarize our current understanding of abnormal repeat RNA metabolism and repeat-associated non-AUG translation in C9orf72 frontotemporal lobar degeneration/ALS. For repeat RNA metabolism, we specifically focus on the role of hnRNPA3, the repeat RNA-binding protein, and the EXOSC10/RNA exosome complex, an intracellular RNA-degrading enzyme. In addition, the mechanism of repeat-associated non-AUG translation inhibition via TMPyP4, a repeat RNA-binding compound, is discussed.

The carboxyl termini of RAN translated GGGGCC nucleotide repeat expansions modulate toxicity in models of ALS/FTD.

An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5' untranslated region (UTR) of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5' UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus (C-terminus) in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR C-terminus was observed when repeats were expressed in rodent neurons. The presence of the native C-termini across all three reading frames was partly protective. Taken together, these findings suggest that C-terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.