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High‐throughput identification of RNA nuclear enrichment sequences
Author(s) -
Shukla Chinmay J,
McCorkindale Alexandra L,
Gerhardinger Chiara,
Korthauer Keegan D,
Cabili Moran N,
Shechner David M,
Irizarry Rafael A,
Maass Philipp G,
Rinn John L
Publication year - 2018
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.201798452
Subject(s) - biology , identification (biology) , rna , throughput , computational biology , genetics , microbiology and biotechnology , gene , telecommunications , botany , computer science , wireless
Abstract In the post‐genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNA s (lnc RNA s), 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 lnc RNA s. We examined a pool of 11,969 oligos densely tiling 38 human lnc RNA s 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.