Subcellular profiling of macrophage long non-coding RNAs

Recent advances in next generation sequencing have improved human genome annotations and revealed thousands of previosly unknown long non-coding RNA loci. Here, we characterized immune-responsive long non-coding RNAs (lncRNAs) and determined their subcellular localization and co-sedimentation with protein complexes in primary human macrophages. To this end, we profiled LPS-responsive lncRNAs, isolated cytoplasmic and nuclear RNA fractions from mock- and LPS-treated cells and seperated cell lysates on 10-60 % glycerol gradients, followed by gradient fractionation. All samples were subjected to RNA-Seq analysis. LPS-responsive lncRNAs were found to be mostly cytoplasmic. Glycerol gradient datasets revealed that a substantial fraction of LPS-responsive lncRNAs, similar to mRNAs, co-sediments with ribosomal RNAs and also ribosomal proteins, as confirmed by mass-spectrometry analysis. LncRNAs not co-sedimenting with ribosomes displayed a highly heterogenous gradient distriubtion. Among these truly non-coding RNAs we identified lncRNA MaIL1 as a novel element of the macrophage TLR-TRIF signaling pathway contributing to antibacterial defense. Expression profiles of poly(A)-RNA from primary human macrophages (+/- LPS treatment) or subcellular macrophage fractions (+/- LPS treatment) were recorded in experimental replicates. RNA profiles of gradient fractionated lysates from LPS-stimulated macrophages were recorded without prior RNA enrichment or depletion steps. For MaIL1 and control RNAi experiments each RNA-Seq dataset (poly(A)-RNA) represents an equimolar pool of RNA from two independent experiments.

下一代测序技术（next generation sequencing）的近期进展完善了人类基因组注释（human genome annotations），并发现了数千个此前未知的长链非编码RNA（long non-coding RNA, lncRNA）基因座。本研究对免疫应答性长链非编码RNA（lncRNA）开展了特征分析，明确了其在原代人巨噬细胞（primary human macrophages）中的亚细胞定位，以及与蛋白质复合物的共沉降特性。为此，我们对脂多糖（Lipopolysaccharide, LPS）应答性lncRNA进行了表达谱分析：从模拟处理组与LPS处理组细胞中分离细胞质与细胞核RNA组分，利用10%~60%甘油梯度对细胞裂解液进行分离，随后完成梯度分级分离。所有样本均接受了RNA测序（RNA-Seq）分析。研究结果显示，LPS应答性lncRNA大多定位于细胞质。甘油梯度数据集表明，与信使RNA（messenger RNA, mRNA）类似，大部分LPS应答性lncRNA可与核糖体RNA及核糖体蛋白发生共沉降，该结论经质谱（mass spectrometry）分析得以验证。不与核糖体共沉降的lncRNA则呈现出高度异质性的梯度分布。在这些真正的非编码RNA中，我们鉴定出lncRNA MaIL1是巨噬细胞TLR-TRIF信号通路的新型调控元件，可参与抗菌防御过程。本研究通过生物学重复记录了两类样本的聚腺苷酸化RNA（poly(A)-RNA）表达谱：一是经或未经LPS处理的原代人巨噬细胞，二是经或未经LPS处理的巨噬细胞亚细胞组分。针对LPS刺激的巨噬细胞的梯度分级裂解液，本研究在未预先开展RNA富集或去除操作的前提下，记录了其RNA表达谱。针对MaIL1及对照RNA干扰（RNA interference, RNAi）实验，每个RNA测序数据集（聚腺苷酸化RNA）均为两次独立实验所得RNA的等摩尔混合池。