High-throughput identification of RNA nuclear enrichment sequences

EMBO J. 2018 Mar 15;37(6):e98452. doi: 10.15252/embj.201798452. Epub 2018 Jan 15.


In the post-genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever-growing catalogs require high-throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA-based functionality. We applied MPRNA to identify RNA-based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine-rich motif. These nuclear enrichment sequences are highly conserved and over-represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single-molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA-based functionalities.

Keywords: RNA; de novo inference of regions; high‐throughput reporter assay; lncRNA; nuclear localization.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Nucleus / genetics
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • In Situ Hybridization, Fluorescence
  • RNA, Long Noncoding / genetics*
  • Sequence Analysis, RNA


  • RNA, Long Noncoding