Solution structure of poly(UG) RNA reveals a new fold that drives gene silencing

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The human genome is extraordinarily abundant in polymorphic (GT)n microsatellite sequences, which code for poly(UG) dinucleotide repeats in RNA (hereafter, “pUGs”). Expansion of pUG sequences have been correlated with diseases such as cancer and cystic fibrosis. In contrast, C. elegans lacks genomic pUGs but possesses a ribonucleotidyl transferase that can add “pUG tails” to RNA 3’ ends. These pUG tails turn RNAs into potent vectors for gene silencing. We therefore hypothesized that pUGs may form RNA structures responsible for gene silencing and possibly, human disease. We report the solution structure of the pUG sequence (GU)12 using solution NMR in combination with SAXS-WAXS scattering data at 0.75 Å r.m.s.d. The RNA folds into a newly identified single-stranded quadruplex characterized by its unique left-handed structure which we define as the “pUG fold”. This structure has four-fold symmetry with non-sequential stacking of 3 G quartets and a U quartet. Other U nucleotides are either budged or in propeller loops. Interestingly, the backbone presents alternate inversions at the quartets, a feature that closely resembles Z-RNA. NMR H/D exchange measurements indicate the pUG fold is kinetically stable for days. We also determined the x-ray crystal structure of (GU)12 bound to the quadruplex ligand N-methyl meosporphyrin IX (NMM). The structures have an r.m.s.d of 1.7 Å, indicating that NMM docks to a pre-folded pUG structure. NMR revealed features of the RNA that were poorly resolved by the electron density maps. The importance of the pUG fold for gene silencing in C. elegans was demonstrated by incorporation of the modified nucleotide 7-deaza guanosine into pUG tailed RNAs. The modified nucleotides disrupt RNA folding and potently inhibit gene silencing. We find ∼20,000 pUG sequences in human genes, suggesting roles beyond gene silencing.