Differential Impact of the HEN1 Homolog HENN-1 on 21U and 26G RNAs in the Germline of Caenorhabditis elegans

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（RNA interference, RNAi）相关通路通过将Argonaute（Ago）蛋白序列特异性招募至mRNA靶分子来调控基因活性。该过程的序列特异性来源于Ago蛋白结合的小分子RNA（small RNA, sRNA）辅助因子。部分通路内的sRNA分子稳定性，部分通过Hen1介导的3'端甲基化过程实现调控。本研究针对秀丽隐杆线虫（Caenorhabditis elegans）的RNA甲基转移酶同源物HENN-1，阐明其对该线虫不同RNAi通路的调控效应。我们揭示了HENN-1对两种已知依赖甲基化sRNA的通路——26G通路与21U通路——的差异化调控作用。令人意外的是，在生殖系中，21U RNA（即秀丽隐杆线虫的piRNA，PIWI-interacting RNA）的稳定性仅因甲基化缺失受到轻度影响；且人工引入21U靶RNA并不会进一步加剧未甲基化21U RNA的不稳定状态。与之相反，大多数26G RNA的稳定性会出现下降，并在HENN-1缺失时表现出3'端尿苷化频率升高的特征。在26G RNA这一类别中，我们发现仅ERGO-1结合的26G RNA会被HENN-1修饰，而ALG-3/ALG-4结合的26G RNA则不会发生该修饰。对henn-1突变体的全局基因表达分析显示出轻度的调控效应，包括大量生殖系表达基因的下调。我们的数据表明，除HENN-1缺失导致26G RNA水平降低对基因表达产生的直接效应外，绝大多数对全局基因表达的影响均为间接效应。本研究进一步完善了我们对秀丽隐杆线虫内源性RNAi通路以及Hen1同源酶在这些通路中所发挥作用的认知。