Robust partitioning of microRNA targets from downstream regulatory changes [miRNA-seq]. Robust partitioning of microRNA targets from downstream regulatory changes [miRNA-seq]

The biological impact of microRNAs is determined by their targets, and robustly identifying direct miRNA targets remains challenging. Existing methods suffer from high false-positive rates and are unable to effectively differentiate direct miRNA targets from downstream regulatory changes. Here, we present a simple approach to deconvolute post-transcriptional and transcriptional changes using PRO-seq with RNA-seq. In combination, these methods allow us to systematically profile the regulatory impact of a miRNA. We refer to this approach as CARP: Combined Analysis of RNA-seq and PRO-seq. We apply CARP to multiple miRNAs and show that it robustly distinguishes direct targets from downstream changes, while greatly reducing false positives. We validate our approach using Argonaute eCLIP-seq and ribosome profiling, demonstrating that CARP defines a comprehensive repertoire of targets. We identify miRNA-specific activity of target sites within the coding region. CARP facilitates the dissection of complex changes in gene regulatory networks triggered by miRNAs and identification of transcription factors that underlie downstream regulatory changes. Given the robustness of the approach, CARP is particularly suitable for dissecting miRNA regulatory networks in vivo. Overall design: MicroRNA profiling of HEK293 cells with and without specific miRNAs by high-throughput sequencing using Illumina NextSeq500.

微小RNA（microRNAs，miRNA）的生物学功能由其靶标决定，而稳健鉴定直接miRNA靶标仍具挑战性。现有方法存在假阳性率高的问题，且无法有效区分直接miRNA靶标与下游调控变化。本研究提出一种结合PRO-seq与RNA-seq的简易分析策略，可解析转录后与转录水平的基因表达变化。二者联用可系统性分析miRNA的调控效应。我们将该方法命名为CARP：RNA-seq与PRO-seq联合分析（Combined Analysis of RNA-seq and PRO-seq）。我们将CARP应用于多种miRNA的分析，结果表明该方法可稳健区分直接靶标与下游调控变化，并大幅降低假阳性率。我们通过Argonaute蛋白eCLIP-seq与核糖体谱分析（ribosome profiling）验证了该方法，证实CARP可鉴定出一套完整的miRNA靶标全集。本研究还鉴定出编码区内的靶标位点具有miRNA特异性的调控活性。CARP可助力解析miRNA诱导的基因调控网络中的复杂变化，并鉴定出介导下游调控变化的转录因子。鉴于该方法的稳健性，CARP尤其适用于体内miRNA调控网络的解析。实验整体设计：利用Illumina NextSeq500平台开展高通量测序，对转染特异性miRNA与未转染的HEK293细胞进行miRNA表达谱分析。