Knittel Thiago L, Montgomery Brooke E, Tate Alex J, Deihl Ennis W, Nawrocki Anastasia S, Hoerndli Frederic J, Montgomery Taiowa A
Department of Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
Genome Res. 2024 Dec 23;34(12):2203-2216. doi: 10.1101/gr.279083.124.
Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by Dicer and associate with Argonautes to direct RNA silencing. In , 22G-RNAs and 26G-RNAs are often referred to as siRNAs but display distinct characteristics. For example, 22G-RNAs do not originate from dsRNA and do not depend on Dicer, whereas 26G-RNAs require Dicer but derive from an atypical RNA duplex and are produced exclusively antisense to their messenger RNA (mRNA) templates. To identify canonical siRNAs in , we first characterized the siRNAs produced via the exogenous RNA interference (RNAi) pathway. During RNAi, dsRNA is processed into ∼23 nt duplexes with ∼2 nt, 3'-overhangs, ultimately yielding siRNAs devoid of 5'G-containing sequences that bind with high affinity to the Argonaute RDE-1, but also to the microRNA (miRNA) pathway Argonaute, ALG-1. Using these characteristics, we searched for their endogenous counterparts and identified thousands of endogenous loci representing dozens of unique elements that give rise to mostly low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, putative coding genes, pseudogenes, noncoding RNAs, and unannotated features, many of which adopt hairpin (hp) structures reminiscent of the hpRNA/RNAi pathway in flies and mice. RDE-1 competes with other Argonautes for binding to 23H-RNAs. When RDE-1 is depleted, these siRNAs are enriched in ALG-1 and ALG-2 complexes. Our results expand the known repertoire of small RNAs and their Argonaute interactors, and demonstrate that key features of the endogenous siRNA pathway are relatively unchanged in animals.
经典的小干扰RNA（siRNA）由Dicer从双链RNA（dsRNA）加工而来，并与AGO蛋白结合以指导RNA沉默。在秀丽隐杆线虫中，22G-RNA和26G-RNA常被称为siRNA，但具有不同的特征。例如，22G-RNA并非源自dsRNA，也不依赖Dicer，而26G-RNA需要Dicer，但源自非典型RNA双链体，并且仅以与其信使RNA（mRNA）模板反义的方式产生。为了在秀丽隐杆线虫中鉴定经典siRNA，我们首先对通过外源RNA干扰（RNAi）途径产生的siRNA进行了表征。在RNAi过程中，dsRNA被加工成约23个核苷酸的双链体，带有约2个核苷酸的3'端突出，最终产生不含与AGO蛋白RDE-1高亲和力结合的含5'G序列的siRNA，同时也与微小RNA（miRNA）途径的AGO蛋白ALG-1结合。利用这些特征，我们寻找它们的内源性对应物，并鉴定出数千个内源性位点，这些位点代表了数十个独特元件，产生的大多是低至中等水平的siRNA，称为23H-RNA。这些位点包括重复元件、推定的编码基因、假基因、非编码RNA和未注释的特征，其中许多采用发夹（hp）结构，让人联想到果蝇和小鼠中的hpRNA/RNAi途径。RDE-1与其他AGO蛋白竞争结合23H-RNA。当RDE-1缺失时，这些siRNA在ALG-1和ALG-2复合物中富集。我们的结果扩展了已知的小RNA及其AGO相互作用蛋白的种类，并证明内源性siRNA途径的关键特征在动物中相对不变。
