High‐throughput identification of RNA nuclear enrichment sequences

• Chinmay J Shukla ^[1] ; Alexandra L McCorkindale ^[2] ; Chiara Gerhardinger ^[1] ; Keegan D Korthauer ^[3] ; Moran N Cabili ^[4] ; David M Shechner ^[1] ; Rafael A Irizarry ^[3] ; Philipp G Maass ^[1] ; John L Rinn ^[5]
  1. [1] Harvard University

     Harvard University

     City of Cambridge, Estados Unidos

     
  2. [2] 1 Department of Stem Cell and Regenerative Biology Harvard University Cambridge MA USA; 5 Berlin Institute for Medical Systems Biology Max Delbrück Center for Molecular Medicine Berlin Germany
  3. [3] 3 Department of Biostatistics and Computational Biology Dana‐Farber Cancer Institute Boston MA USA; 6 Department of Biostatistics Harvard T.H. Chan School of Public Health Boston MA USA
  4. [4] 2 Broad Institute of MIT and Harvard Cambridge MA USA
  5. [5] 1 Department of Stem Cell and Regenerative Biology Harvard University Cambridge MA USA; 2 Broad Institute of MIT and Harvard Cambridge MA USA; 7 Department of Pathology Beth Israel Deaconess Medical Center Boston MA USA; 8Present address: Department of Biochemistry University of Colorado BioFrontiers Boulder CO USA

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• Localización: EMBO journal: European Molecular Biology Organization, ISSN 0261-4189, Vol. 37, Nº. 6, 2018
• Idioma: inglés
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  • 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.