Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
RNA. 2010 Apr;16(4):673-95. doi: 10.1261/rna.2000810. Epub 2010 Feb 24.
Competition between mammalian RNAi-related gene silencing pathways is well documented. It is therefore important to identify all classes of small RNAs to determine their relationship with RNAi and how they affect each other functionally. Here, we identify two types of 5'-phosphate, 3'-hydroxylated human tRNA-derived small RNAs (tsRNAs). tsRNAs differ from microRNAs in being essentially restricted to the cytoplasm and in associating with Argonaute proteins, but not MOV10. The first type belongs to a previously predicted Dicer-dependent class of small RNAs that we find can modestly down-regulate target genes in trans. The 5' end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 3' end resulted from transcription termination by RNA polymerase III. Consistent with their preferential association with the nonslicing Argonautes 3 and 4, canonical gene silencing activity was not observed for type II tsRNAs. The addition, however, of an oligonucleotide that was sense to the reporter gene, but antisense to an overexpressed version of the type II tsRNA, triggered robust, >80% gene silencing. This correlated with the redirection of the thus reconstituted fully duplexed double-stranded RNA into Argonaute 2, whereas Argonautes 3 and 4 were skewed toward less structured small RNAs, particularly single-strand RNAs. We observed that the modulation of tsRNA levels had minor effects on the abundance of microRNAs, but more pronounced changes in the silencing activities of both microRNAs and siRNAs. These findings support that tsRNAs are involved in the global control of small RNA silencing through differential Argonaute association, suggesting that small RNA-mediated gene regulation may be even more finely regulated than previously realized.
哺乳动物 RNA 干扰相关基因沉默途径的竞争已有充分的文献记载。因此，确定所有小 RNA 类别以确定它们与 RNAi 的关系以及它们如何在功能上相互影响非常重要。在这里，我们鉴定了两种类型的 5'-磷酸化、3'-羟基化的人 tRNA 衍生的小 RNA（tsRNAs）。tsRNAs 与 microRNAs 的不同之处在于它们本质上局限于细胞质，并与 Argonaute 蛋白结合，而不是与 MOV10 结合。第一种类型属于以前预测的依赖 Dicer 的小 RNA 类，我们发现它可以适度下调靶基因的转录。II 型 tsRNA 的 5' 端是由 tRNA 基因下游的 RNaseZ 切割产生的，而 3' 端则是由 RNA 聚合酶 III 的转录终止产生的。与它们优先与非切割性 Argonautes 3 和 4 结合一致，我们没有观察到 II 型 tsRNA 的典型基因沉默活性。然而，添加与报告基因有意义但与 II 型 tsRNA 的过表达版本反义的寡核苷酸，会引发强烈的、超过 80%的基因沉默。这与重新构成的完全双链 RNA 被定向到 Argonaute 2 相关，而 Argonautes 3 和 4 则偏向于较少结构的小 RNA，特别是单链 RNA。我们观察到 tsRNA 水平的调节对 microRNAs 的丰度只有较小的影响，但对 microRNAs 和 siRNAs 的沉默活性都有更明显的变化。这些发现表明，tsRNAs 通过差异的 Argonaute 结合参与小 RNA 沉默的全局控制，这表明小 RNA 介导的基因调控可能比以前认识到的更加精细。
