Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity (hESC). Homo sapiens

Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called “isomiRs” adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes–PAPD1, ZCCHC6 and TUT1–have not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. Overall design: RNA was isolated from undifferentiated or differentiated H1 human embryonic cells as previously described (Bar et al, 2008). Two biological replicates of undifferentiated and differentiated cell RNA was profiled on the NanoString nCounter miRNA expression assay adapted to discriminate miRNA 3' variants.

通过在3'端添加核苷酸修饰微小RNA（microRNA，miRNA）序列以生成所谓的“异miR（isomiRs）”，这一过程增加了miRNA功能的复杂性；近期研究表明，3'端修饰可影响miRNA的稳定性及其靶基因抑制效率。
本研究证实，miRNA的3'端修饰是一类生理性且普遍存在的转录后事件，其对特定miRNA具有选择性，且在从秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）到人类的多个物种中均有存在。这类修饰主要源于腺苷酸化与尿苷酸化过程，可在多种组织类型、疾病状态及发育阶段中被检测到。
为实现3'端核苷酸添加事件的定量表征，我们开发并验证了一种基于NanoString Technologies的nCounter平台的新型检测方法。针对部分miRNA，其修饰频率会因细胞分化等过程发生改变，这提示3'端修饰是一个受生物学调控的过程。
为探究3'端核苷酸添加的作用机制，我们借助RNA干扰（RNA interference，RNAi）技术，对人类细胞中8个候选的miRNA核苷酸转移酶开展了3'端miRNA修饰活性筛选。研究发现，包括PAPD1、PAPD4、PAPD5、ZCCHC6、ZCCHC11及TUT1在内的多种酶，可通过miRNA特异性的方式调控miRNA的3'端核苷酸添加。其中PAPD1、ZCCHC6及TUT1这3种酶，此前尚未被发现具有miRNA修饰功能。
综上，我们的研究结果表明，下一代小RNA测序（next generation small RNA sequencing）数据中观测到的3'端修饰是一类具有生物学相关性的过程，并明确了可用于在体内调控miRNA活性的潜在酶学机制，为开发新型体内miRNA活性调控策略提供了方向。
整体实验设计：如既往研究所述（Bar等，2008），我们从未分化或已分化的H1人类胚胎细胞中提取总RNA。未分化与已分化细胞的RNA各设置两个生物学重复样本，采用经改造可区分miRNA 3'端变体的NanoString nCounter miRNA表达检测试剂盒完成表达谱分析。