An Atypical RNA Quadruplex Marks RNAs as Vectors for Gene Silencing

Abstract

The addition of poly(UG) ("pUG") repeats to 3' termini of mRNAs drives gene silencing and trans-generational epigenetic inheritance in the metazoan C. elegans. pUG tails promote silencing by recruiting an RNA-dependent RNA Polymerase (RdRP) that synthesizes small interfering (si)RNAs. Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4)2 structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. Its biological importance is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific N7-deaza RNA substitutions that do not adopt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA secondary structure motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in non-coding regions of human RNAs, suggesting the fold likely has biological roles beyond gene silencing.