ABSTRACT

microRNAs (miRNAs) are small non-coding RNAs that play critical roles in gene regulation. The presence of miRNAs in extracellular biofluids is increasingly recognized. However, most previous characterization of extracellular miRNAs focused on their overall expression levels. Alternative sequence isoforms and modifications of miRNAs were rarely considered in the extracellular space. Here, we developed a highly accurate bioinformatic method, called miNTA, to identify 3’ non-templated additions (NTAs) of miRNAs using small RNA-sequencing data. Using miNTA, we conducted an in-depth analysis of miRNA 3’ NTA profiles in 1047 extracellular RNA-sequencing data sets of 4 types of biofluids. This analysis identified hundreds of miRNAs with 3’ uridylation or adenylation, with the former being more prevalent. Among these miRNAs, up to 53% (22%) had an average 3’ uridylation (adenylation) level of at least 10% in a specific biofluid. Strikingly, we found that 3’ uridylation levels enabled segregation of different types of biofluids, more effectively than overall miRNA expression levels. This observation suggests that 3’ NTA levels possess fluid-specific information relatively robust to batch effects. In addition, we observed that extracellular miRNAs with 3’ uridylations are enriched in processes related to angiogenesis, apoptosis, and inflammatory response, and this type of modification may stabilize base-pairing between miRNAs and their target genes. Together, our study provides a comprehensive landscape of miRNA NTAs in human biofluids, which paves way for further biomarker discoveries. The insights generated in our work built a foundation for future functional, mechanistic, and translational discoveries.

摘要

microRNA (miRNA) 是一种小型非编码 RNA，在基因调控中发挥着关键作用。人们越来越认识到细胞外生物液中存在 miRNA。然而，之前大多数细胞外 miRNA 的表征都集中在它们的整体表达水平上。在细胞外空间中很少考虑 miRNA 的替代序列亚型和修饰。在这里，我们开发了一种高度准确的生物信息学方法，称为 miNTA，使用小 RNA 测序数据来识别 miRNA 的 3' 非模板添加 (NTA)。使用 miNTA，我们对 4 种生物体液的 1047 个细胞外 RNA 测序数据集中的 miRNA 3' NTA 图谱进行了深入分析。该分析鉴定出数百个具有 3' 尿苷酸化或腺苷酸化的 miRNA，其中前者更为普遍。在这些 miRNA 中，高达 53% (22%) 在特定生物液中的平均 3' 尿苷化（腺苷酸化）水平至少为 10%。引人注目的是，我们发现 3' 尿苷化水平能够比整体 miRNA 表达水平更有效地分离不同类型的生物流体。这一观察结果表明，3' NTA 水平拥有相对稳定的批次效应的流体特异性信息。此外，我们观察到具有3'尿苷化的细胞外miRNA在与血管生成、细胞凋亡和炎症反应相关的过程中富集，并且这种类型的修饰可以稳定miRNA与其靶基因之间的碱基配对。总之，我们的研究提供了人类生物体液中 miRNA NTA 的全面概况，这为进一步发现生物标志物铺平了道路。我们工作中产生的见解为未来的功能、机械和转化发现奠定了基础。