A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming [small RNA-seq]

MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to broadly repress mRNA targets. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter targets with extensive complementarity, which can trigger a process termed target-directed microRNA degradation (TDMD). Prevailing models of TDMD invoke miRNA tailing and trimming as an essential step in the decay mechanism. Here, a genome-wide screen revealed a novel cullin-RING ubiquitin ligase, which we named the DECAY complex, that mediates TDMD. The DECAY complex interacts with AGO proteins, mediates TDMD induced by multiple transcripts, and does not require tailing and trimming to elicit miRNA turnover. Based upon these findings, we propose a model in which the DECAY complex mediates TDMD by promoting proteasomal decay of miRNA-containing complexes. Overall design: Total RNA was isolated from biological triplicates of each cell line using the miRNeasy Mini RNA isolation kit (QIAGEN). Library preparation and next generation sequencing were performed by DNA Link using NEB Next Small RNA Library Prep for Illumina (New England BioLabs).

微小RNA（microRNAs，miRNAs）可与Argonaute（AGO）蛋白协同作用，广泛抑制mRNA靶标。在结合AGO蛋白后，miRNAs通常展现出缓慢的周转速率。但存在一个重要例外：当miRNAs与具有广泛互补性的靶标结合时，会触发一种被称为靶标导向微小RNA降解（target-directed microRNA degradation, TDMD）的过程。当前主流的TDMD模型认为，miRNA的尾端添加与修剪是该降解机制的核心步骤。本研究通过全基因组筛选发现了一种新型cullin-RING泛素连接酶，我们将其命名为DECAY复合物，该复合物可介导TDMD过程。DECAY复合物可与AGO蛋白相互作用，介导由多种转录本诱导的TDMD过程，且无需通过尾端添加与修剪即可引发miRNA的周转。基于上述发现，我们提出了一种新模型：DECAY复合物通过促进包含miRNA的复合物的蛋白酶体降解来介导TDMD过程。
整体实验设计：使用miRNeasy迷你RNA提取试剂盒（QIAGEN）从每个细胞系的三份生物学重复样本中提取总RNA。文库制备与下一代测序由DNA Link公司使用NEBNext Small RNA Library Prep for Illumina（New England BioLabs）完成。