Structural characterization of NORAD reveals a stabilizing role of spacers and two new repeat units

Long non-coding RNAs (IncRNAs) can perform a variety of key cellular functions by interacting with proteins and other RNAs. Recent studies have shown that the function of IncRNAS are largely mediated by their structures. However, our structural knowledge for most IncRNAS is limited to sequence-based computational predictions. Non-coding RNA activated by DNA damage (NORAD) is an atypical IncRNA due to its abundant expression and high sequence conservation. NORAD regulates genomic stability by interacting with proteins and microRNAs. Previous sequence-based characterization has identified a modular organization of NORAD composed of several NORAD repeat units (NRUs). These units comprise the protein-binding elements and are separated by regular spacers of unknown function. Here, we experimentally determine for the first time the secondary structure of NORAD using the nextPARS approach. Our results suggest that the spacer regions provide structural stability to NRUs. Furthermore, we uncover two previously-unreported NRUs, and determine the core structural motifs conserved across NRUs. Overall, these findings will help to elucidate the function and evolution of NORAD.