Structural and biochemical insights into RNA unfolding by a chaperone machine

Although ATP-independent chaperones assist RNA folding, the mechanisms by which they function remain elusive. Here, we demonstrate how two chaperones collaborate to unfold misfolded noncoding RNAs (ncRNAs). Ro60 is a ring that binds the ends of misfolded ncRNAs in its cavity, while La binds many nascent ncRNAs, stabilizing them and assisting correct folding. Using cryoelectron microscopy to resolve the structure of a misfolded RNA complexed with Ro60 and La, we show that La cradles the Ro60 RNP, with its N-terminal domain binding the RNA 3' end after it passes through the Ro60 cavity, while its C-terminal domain interacts with the RNA body. Using SHAPE-MaP, we show that La and Ro60 function synergistically to disrupt weak structural elements. As the RNAs bound by Ro60 and La include both ncRNA precursors and RNAs with oligouridine tails, La- and Ro60-mediated RNA unfolding may both promote correct folding and assist misfolded RNA degradation. Overall design: From RO60-/- human HaCaT cells expressing Ro60 fused to twin-strep-tag, Ro60/La RNP bound RNAs were purified by strep-tactin pulldown followed by immunoprecipitation of La. cDNAs were made after Ro60/La RNP bound RNAs were extracted and ligated to 3' adapter, followed by library construction and sequencing.