Abstract
Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Particularly enigmatic lncRNAs are those that are exported to the cytoplasm, including NORAD—an abundant and highly conserved cytoplasmic lncRNA. Here we show that most of the sequence of NORAD is comprised of repetitive units that together contain at least 17 functional binding sites for the two mammalian Pumilio homologues. Through binding to PUM1 and PUM2, NORAD modulates the mRNA levels of their targets, which are enriched for genes involved in chromosome segregation during cell division. Our results suggest that some cytoplasmic lncRNAs function by modulating the activities of RNA-binding proteins, an activity which positions them at key junctions of cellular signalling pathways.
Highlights
Thousands of long noncoding RNA genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure
Using single-molecule in situ hybridization[19] in U2OS cells, we found that NORAD localizes almost exclusively to the cytoplasm (Fig. 1c and Supplementary Fig. 2) and similar cytoplasmic enrichment is observed in other cells lines (Fig. 1d)
The number of NORAD copies expressed in a cell is B80 based on the RPKM data and 68±8 based on the smFISH experiments that we have performed on U2OS cells, with 94% of NORAD copies located in the cytoplasm and 6% in the nucleus
Summary
Thousands of long noncoding RNA (lncRNA) genes are encoded in the human genome, and hundreds of them are evolutionarily conserved, but their functions and modes of action remain largely obscure. Most lncRNAs remain poorly characterized, and the few well-studied examples consist of lncRNAs that act in the nucleus to regulate the activity of loci found in cis to their sites of transcription[3]. These include the XIST lncRNA, a key component of the X-inactivation pathway, and lncRNAs that are instrumental for imprinting processes, such as AIRN4. Two lncRNAs that are spliced into circular forms were shown to act in the cytoplasm by binding Argonaute proteins (in one case, through B70 binding sites for a miR-7 microRNA7) and act as sponges that modulate microRNA-mediated repression[7,8] Such examples are probably rare, as few circRNAs and few lncRNAs contain multiple canonical microRNA-binding sites[9]. Using a luciferase reporter system we show that this modulation depends on the canonical Pumilio binding sites
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