Dis3l2-Mediated RNA Surveillance and Extracellular Vesicle Packaging Prevent Innate Immune Activation by Aberrant Cellular RNAs [RNA-seq]

Although most efforts to understand the functions of RNAs packaged in extracellular vesicles (EVs) have focused on mRNAs and miRNAs, recent studies using less-biased sequencing techniques have revealed that tRNAs and other noncoding RNAs (ncRNAs) are far more abundant. However, the extent to which EV ncRNAs resemble overall cellular RNAs and the benefits to host cells of packaging them into EVs remain unknown. Here, we purified EVs from the culture media of mouse and human cells and characterized their RNA components using high throughput sequencing and Northern blotting. We report that EVs are enriched for numerous aberrant ncRNAs, many of which contain oligouridine tails, a modification associated with degradation by Dis3l2, a cytosolic exoribonuclease that degrades defective structured ncRNAs. Both the numbers of EVs released and the fractions of tailed RNAs in EVs increase on Dis3l2 depletion, indicating that packaging of aberrant ncRNAs into EVs occurs in competition with Dis3l2 decay. Upregulation of multiple type I interferon stimulated genes (ISGs) occurs on Dis3l2 depletion, and cells treated with a neutral sphingomyelinase inhibitor that reduces exosome biogenesis also show moderate ISG upregulation, an effect that is enhanced in Dis3l2-depleted cells and accompanied by the accumulation of aberrant ncRNAs in cellular RNA. Thus, Dis3l2 degradation and packaging of aberrant RNAs into vesicles may prevent these RNAs from activating innate immune sensors and triggering an interferon response. Overall design: Total RNA-seq profiling of RNA purified from RAW 264.7 cells expressing a non-silencing shRNA (shNS) and Dis3l2 KD cells (shDis3l2).

尽管学界对包装于细胞外囊泡（extracellular vesicles, EVs）中的RNA功能的研究多聚焦于信使RNA（mRNA）与微小RNA（miRNA），但近期采用低偏好性测序技术开展的研究显示，转运RNA（tRNA）及其他非编码RNA（ncRNA）的丰度要高得多。然而，细胞外囊泡非编码RNA（EV ncRNA）与整体细胞RNA的相似程度，以及将其包装入细胞外囊泡对宿主细胞的益处，目前仍不明确。本研究从小鼠及人类细胞的培养基中纯化得到细胞外囊泡，并通过高通量测序（high throughput sequencing）与Northern印迹（Northern blotting）对其RNA组分进行了表征。本研究发现，细胞外囊泡中富集了大量异常非编码RNA，其中许多带有寡聚尿苷尾——这种修饰与Dis3l2介导的降解相关；Dis3l2是一种可降解结构异常非编码RNA的胞质外核糖核酸酶。当Dis3l2表达被敲低时，细胞释放的细胞外囊泡数量以及囊泡中带尾RNA的占比均会升高，这表明异常非编码RNA被包装入细胞外囊泡的过程，与Dis3l2介导的降解存在竞争关系。当Dis3l2被敲低时，多种I型干扰素刺激基因（ISG）的表达会上调；用可抑制外泌体生物发生的中性鞘磷脂酶抑制剂处理细胞，也会使ISG表达出现中等程度的上调，且该效应在Dis3l2敲低的细胞中会增强，同时伴随细胞RNA中异常非编码RNA的积累。由此可见，Dis3l2介导的降解以及将异常RNA包装入囊泡的过程，或可阻止这些RNA激活先天免疫感受器并触发干扰素应答。
整体实验设计：对表达非沉默短发夹RNA（shRNA）的RAW 264.7细胞以及Dis3l2敲低（KD）细胞（shDis3l2）中纯化得到的RNA进行总RNA测序（Total RNA-seq）分析。