Kornienko Aleksandra E, Vlatkovic Irena, Neesen Jürgen, Barlow Denise P, Pauler Florian M
a CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH BT 25.3 , 1090 Vienna , Austria.
b Institute of Medical Genetics, Medical University of Vienna, Währingerstrasse 10 , 1090 Vienna , Austria.
RNA Biol. 2016;13(2):196-220. doi: 10.1080/15476286.2015.1110676.
Many thousand long non-coding (lnc) RNAs are mapped in the human genome. Time consuming studies using reverse genetic approaches by post-transcriptional knock-down or genetic modification of the locus demonstrated diverse biological functions for a few of these transcripts. The Human Gene Trap Mutant Collection in haploid KBM7 cells is a ready-to-use tool for studying protein-coding gene function. As lncRNAs show remarkable differences in RNA biology compared to protein-coding genes, it is unclear if this gene trap collection is useful for functional analysis of lncRNAs. Here we use the uncharacterized LOC100288798 lncRNA as a model to answer this question. Using public RNA-seq data we show that LOC100288798 is ubiquitously expressed, but inefficiently spliced. The minor spliced LOC100288798 isoforms are exported to the cytoplasm, whereas the major unspliced isoform is nuclear localized. This shows that LOC100288798 RNA biology differs markedly from typical mRNAs. De novo assembly from RNA-seq data suggests that LOC100288798 extends 289kb beyond its annotated 3' end and overlaps the downstream SLC38A4 gene. Three cell lines with independent gene trap insertions in LOC100288798 were available from the KBM7 gene trap collection. RT-qPCR and RNA-seq confirmed successful lncRNA truncation and its extended length. Expression analysis from RNA-seq data shows significant deregulation of 41 protein-coding genes upon LOC100288798 truncation. Our data shows that gene trap collections in human haploid cell lines are useful tools to study lncRNAs, and identifies the previously uncharacterized LOC100288798 as a potential gene regulator.
人类基因组中已定位了数千种长链非编码（lnc）RNA。通过转录后敲低或基因座的基因改造，利用反向遗传学方法进行的耗时研究表明，其中一些转录本具有多种生物学功能。单倍体KBM7细胞中的人类基因陷阱突变体文库是研究蛋白质编码基因功能的现成工具。由于lncRNA与蛋白质编码基因相比在RNA生物学方面存在显著差异，尚不清楚该基因陷阱文库是否可用于lncRNA的功能分析。在此，我们以未表征的LOC100288798 lncRNA为模型来回答这个问题。利用公开的RNA测序数据，我们发现LOC100288798广泛表达，但剪接效率低下。少量剪接的LOC100288798异构体被输出到细胞质中，而主要的未剪接异构体则定位于细胞核。这表明LOC100288798的RNA生物学与典型mRNA明显不同。从RNA测序数据进行的从头组装表明，LOC100288798在其注释的3'端之外延伸了289kb，并与下游的SLC38A4基因重叠。从KBM7基因陷阱文库中可获得在LOC100288798中具有独立基因陷阱插入的三种细胞系。RT-qPCR和RNA测序证实了lncRNA的成功截断及其延长的长度。RNA测序数据的表达分析表明，LOC100288798截断后41个蛋白质编码基因发生了显著的失调。我们的数据表明，人类单倍体细胞系中的基因陷阱文库是研究lncRNA的有用工具，并将先前未表征的LOC100288798鉴定为潜在的基因调节因子。
