A human haploid gene trap collection to study lncRNAs with unusual RNA biology

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.

人类基因组中已注释数千种长链非编码RNA（long non-coding RNA，lncRNA）。通过转录后基因敲低或基因座遗传修饰的反向遗传学方法开展的研究耗时良久，目前仅证实其中少数转录本具备多样的生物学功能。单倍体KBM7细胞的人类基因陷阱突变体库是研究蛋白质编码基因功能的现成工具。鉴于lncRNA在RNA生物学特性上与蛋白质编码基因存在显著差异，目前尚不明确该基因陷阱库是否可用于lncRNA的功能分析。本研究以功能未明确的LOC100288798 lncRNA为模型，旨在解答这一问题。借助公共RNA测序（RNA-seq）数据，我们证实LOC100288798呈广谱表达，但剪接效率低下。少量剪接的LOC100288798异构体可被转运至细胞质，而主要的未剪接异构体则定位于细胞核。上述结果表明，LOC100288798的RNA生物学特性与典型mRNA存在显著差异。通过RNA-seq数据的从头组装分析发现，LOC100288798的转录本延伸至其注释3'端下游289kb处，并与下游的SLC38A4基因存在序列重叠。KBM7基因陷阱库中存有3株在LOC100288798位点携带独立基因陷阱插入突变的细胞系。实时定量聚合酶链反应（RT-qPCR）与RNA-seq结果证实，LOC100288798被成功截短，且其转录本确实存在延伸序列。基于RNA-seq数据的表达分析显示，LOC100288798截短后，共有41个蛋白质编码基因的表达发生显著失调。本研究结果表明，人类单倍体细胞系的基因陷阱库可作为研究lncRNA功能的有效工具，同时将此前未表征的LOC100288798鉴定为一种潜在的基因调控因子。