The NORAD lncRNA assembles a topoisomerase complex critical for genome stability [RNA-seq]

Thousands of long non-coding RNAs (lncRNAs) have been identified in the human genome, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen lncRNAs. One specific lncRNA, Non-coding RNA Activated by DNA Damage (NORAD), has recently been shown by genetic deletion to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here, we combine RNA antisense purification (RAP) and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX (an emerging component of the DNA-damage response) and encodes the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex, which we term NORAD-Activated Ribonucleoprotein Complex 1 (NARC1), containing known suppressors of genomic instability: topoisomerase I (TOP1), ALYREF and the PRPF19/CDC5L complex. Cells depleted of NORAD or RBMX display an increased frequency of chromosome segregation errors, reduced replication-fork velocity and altered cell cycle progression phenotypes that are mechanistically linked to TOP1 and PRPF19/CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function and that NORAD is required for the assembly of a previously unknown topoisomerase complex (NARC1) that contributes to maintaining genomic stability. Moreover, we uncover a novel function for lncRNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex. Overall design: We examined gene expression changes and alternative splicing events in wildtype and NORAD depleted cells using RNA sequencing.

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Munschauer M, Nguyen CT, Sirokman K, Hartigan CR, Hogstrom L, Engreitz JM, Ulirsch JC, Fulco CP, Subramanian V, Chen J, Schenone M, Guttman M, Carr SA, Lander ES