A Novel RNA Motif Mediates the Strict Nuclear Localization of a Long Noncoding RNA

Abstract

The ubiquitous presence of long noncoding RNAs (lncRNAs) in eukaryotes points to the importance of understanding how their sequences impact function. As many lncRNAs regulate nuclear events and thus must localize to nuclei, we analyzed the sequence requirements for nuclear localization in an intergenic lncRNA named BORG (BMP2-OP1-responsive gene), which is both spliced and polyadenylated but is strictly localized in nuclei. Subcellular localization of BORG was not dependent on the context or level of its expression or decay but rather depended on the sequence of the mature, spliced transcript. Mutational analyses indicated that nuclear localization of BORG was mediated through a novel RNA motif consisting of the pentamer sequence AGCCC with sequence restrictions at positions −8 (T or A) and −3 (G or C) relative to the first nucleotide of the pentamer. Mutation of the motif to a scrambled sequence resulted in complete loss of nuclear localization, while addition of even a single copy of the motif to a cytoplasmically localized RNA was sufficient to impart nuclear localization. Further, the presence of this motif in other cellular RNAs showed a direct correlation with nuclear localization, suggesting that the motif may act as a general nuclear localization signal for cellular RNAs.

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01673-13.

ACKNOWLEDGMENTS

We thank Maria Hatzoglou, William Schiemann, Edward Stavnezer, and M. L. Nikki Harter for the kind gifts of the pLntx plasmid and HeLa, HEK293, NMuMg, 4T1, and C2C12 cells and Jo Ann Wise, Hua Lou, and Ahmad Khalil for critical reading of the manuscript. We also thank Penny Benchek for advice on statistical analyses and Mahshid Malakootian for help in literature search.

This work was supported by grants from the ALS Therapy Alliance and National Center for Regenerative Medicine and Tech 09-071 from the Ohio Third Frontier Program.

B.Z. and S.V. designed the experiments. B.Z. performed the majority of the experiments and analyzed the data. F.N. performed the computational analyses. L.G. participated in analysis of fractionated mutant RNAs. F.J. performed the initial characterization of BORG and the in situ hybridization assays, cloned BORG RNA, and participated in making the mutant constructs and stable cell lines. B.Z. and S.V. prepared the manuscript. S.V. performed the statistical analysis.

We declare that we do not have any competing financial interests.

ADDENDUM IN PROOF

While this paper was in press, analysis of high-throughput sequencing results on RNAs obtained from nuclear and cytoplasmic fractions in mouse cell lines confirmed that the AGCCC motif was enriched in transcripts with a high nuclear/cytoplasmic ratio at a transcriptome-wide level (B. Tian, Rutgers University, personal communication).