Abstract
NORAD is a conserved long noncoding RNA (lncRNA) that is required for genome stability in mammals. NORAD acts as a negative regulator of PUMILIO (PUM) proteins in the cytoplasm, and we previously showed that loss of NORAD or PUM hyperactivity results in genome instability and premature aging in mice (Kopp et al., 2019). Recently, however, it was reported that NORAD regulates genome stability through an interaction with the RNA binding protein RBMX in the nucleus. Here, we addressed the contributions of NORAD:PUM and NORAD:RBMX interactions to genome maintenance by this lncRNA in human cells. Extensive RNA FISH and fractionation experiments established that NORAD localizes predominantly to the cytoplasm with or without DNA damage. Moreover, genetic rescue experiments demonstrated that PUM binding is required for maintenance of genomic stability by NORAD whereas binding of RBMX is dispensable for this function. These data provide an important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance.
Highlights
Long noncoding RNAs have emerged as regulators of diverse biological processes
Initial studies of Noncoding RNA activated by DNA damage (NORAD) revealed that this long noncoding RNA (lncRNA) is required to maintain genomic stability in mammalian cells (Lee et al, 2016), and provided strong evidence that this function is mediated through the ability of NORAD to bind to and negatively regulate PUMILIO RNA binding proteins (PUM1 and PUM2) in the cytoplasm (Lee et al, 2016; Tichon et al, 2016)
A distinct localization pattern was reported based upon RNA fluorescent in situ hybridization (FISH) performed using a commerciallyavailable kit with a proprietary set of oligonucleotide probes that hybridize to an unknown segment of NORAD (Munschauer et al, 2018)
Summary
Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Initial studies of NORAD revealed that this lncRNA is required to maintain genomic stability in mammalian cells (Lee et al, 2016), and provided strong evidence that this function is mediated through the ability of NORAD to bind to and negatively regulate PUMILIO RNA binding proteins (PUM1 and PUM2) in the cytoplasm (Lee et al, 2016; Tichon et al, 2016). Further experiments demonstrated that RBMX is not required for induction of NORAD following DNA damage nor its cytoplasmic localization Together, these studies establish the importance of the NORAD:PUM axis in regulating genomic stability in mammalian cells and provide a foundation for further dissection of the mechanism and physiologic role of this pathway
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