Abstract
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.
Highlights
In the post-genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs, and a cadre of small peptides
In order to identify RNA sequences that drive long noncoding RNAs (lncRNAs) nuclear enrichment, we developed a high-throughput approach for identifying nuclear enrichment elements
We designed a pool of 11,969 153-nt oligos representing 38 lncRNAs with diverse subcellular localization patterns: from single nuclear foci (e.g., XIST, ANRIL, ANCR, PVT1, KCNQ1OT1, FIRRE) to broadly diffuse cytosolic patterns (e.g., NR_024412, NR 033770; Cabili et al, 2015)
Summary
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. 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. We demonstrate the utility of MPRNA for future investigation of RNA-based functionalities
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