Differential Gene Expression following DHX36/G4R1 Knockout Is Associated with G-Quadruplex Content and Cancer.

G-quadruplexes (G4s) are secondary DNA and RNA structures stabilized by positive cations in a central channel formed by stacked tetrads of Hoogsteen base-paired guanines. G4s form from G-rich sequences across the genome, whose biased distribution in regulatory regions points towards a gene-regulatory role. G4s can themselves be regulated by helicases, such as DHX36 (aliases: G4R1 and RHAU), which possess the necessary activity to resolve these stable structures. G4s have been shown to both positively and negatively regulate gene expression when stabilized by ligands, or through the loss of helicase activity. Using DHX36 knockout Jurkat cell lines, we identified widespread, although often subtle, effects on gene expression that are associated with the presence or number of observed G-quadruplexes in promoters or gene regions. Genes that significantly change their expression, particularly those that show a significant increase in RNA abundance under DHX36 knockout, are associated with a range of cellular functions and processes, including numerous transcription factors and oncogenes, and are linked to several cancers. Our work highlights the direct and indirect role of DHX36 in the transcriptome of T-lymphocyte leukemia cells and the potential for DHX36 dysregulation in cancer.

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.