Hubrecht Institute-KNAW and University Medical Centre Utrecht, Utrecht, The Netherlands.
PLoS Genet. 2012;8(7):e1002702. doi: 10.1371/journal.pgen.1002702. Epub 2012 Jul 19.
RNA interference (RNAi)-related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3' ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA-methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3'-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1-bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4-bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.
RNA 干扰 (RNAi) 相关途径通过特定序列招募 Ago 蛋白到 mRNA 靶分子来影响基因活性。该过程的序列特异性源于与 Ago 蛋白结合的小 RNA (sRNA) 辅助因子。在一些途径中，sRNA 分子的稳定性部分受 Hen1 介导的 3'端甲基化调节。在这里，我们描述了秀丽隐杆线虫 HEN1 RNA-甲基转移酶同源物 HENN-1 对该线虫中不同 RNAi 途径的影响。我们揭示了 HENN-1 对两种已知使用甲基化 sRNA 分子的途径的不同影响：26G 和 21U 途径。令人惊讶的是，在生殖系中，21U RNA（秀丽隐杆线虫 piRNAs）的稳定性仅受到甲基化缺失的轻微影响；并且引入人工 21U 靶 RNA 不会进一步使非甲基化的 21U RNA 不稳定。相比之下，大多数 26G RNA 显示出稳定性降低，并且通过显示增加的 3'-尿苷酸化频率对 HENN-1 的缺失做出反应。在 26G RNA 类中，我们发现 ERGO-1 结合的 26G RNA 被 HENN-1 修饰，而 ALG-3/ALG-4 结合的 26G RNA 则不受修饰。对 HENN-1 突变体的全基因表达分析显示出轻微的影响，包括许多生殖系表达基因的下调。我们的数据表明，除了 HENN-1 减少 26G RNA 水平对基因表达的直接影响外，对整体基因表达的大多数影响都是间接的。这些研究进一步完善了我们对秀丽隐杆线虫内源性 RNAi 的理解以及 Hen1 样酶在这些途径中的作用。
