Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
EMBO J. 2018 Mar 15;37(6). 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 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 hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA-based functionalities.
在后基因组时代，已经鉴定出了数千个假定的非编码调控区域，如增强子、启动子、长非编码 RNA（lncRNA）和一系列小肽。这些不断增长的目录需要高通量测定法来大规模测试它们的功能。大规模平行报告基因测定法极大地促进了对非编码 DNA 元件的理解。在这里，我们提出了一种大规模平行 RNA 测定法（MPRNA），可以对 10000 个或更多的 RNA 片段进行基于 RNA 的功能测定。我们应用 MPRNA 来鉴定 lncRNA 中所包含的基于 RNA 的核定位结构域。我们检查了一个由 11969 个 oligo 组成的池，这些 oligo 密集地覆盖了 38 个人类 lncRNA，并将其融合到细胞质转录物上。在细胞分馏和条形码测序后，我们鉴定出了 109 个独特的 RNA 区域，这些区域显著增加了细胞质转录物在细胞核中的含量，包括富含胞嘧啶的基序。这些核富集序列高度保守，在全球核分馏测序中重复出现。重要的是，这些区域中的许多都被单分子 RNA 荧光杂交独立验证。总体而言，我们证明了 MPRNA 在未来研究基于 RNA 的功能方面的效用。
