Systematic Characterization Of Small RNAs Associated With C. Elegans Argonautes

Small RNAs(sRNAs)are RNA molecules that are less than 200 nucleotides in length and play a crucial regulatory role in organisms.Based on production and function mechanisms,sRNAs can be classified into three major categories:microRNAs(miRNAs),small interfering RNAs(siRNAs),and PIWI-interacting RNAs(piRNAs).In C.elegans,siRNAs refer to 22G-RNAs and 26G-RNAs.sRNAs generally play regulatory roles by forming complexes with the corresponding Argonaute,which are core components of the RNA-induced Silencing Complex(RISC).C.elegans has an expanded Argonaute family with 27 Argonaute genes in the genome,20 of which are potentially functional according to WormBase annotation(WS285).To date,limited research has been reported based on the real expression level of Argonaute protein,primarily attributed to the absence of specific antibodies.With the development of gene editing technology,it is highly advantageous to fuse tags to endogenous proteins to facilitate their functional studies.Here,we conduct a systematic study between C.elegans Argonautes and their associated sRNAs by CRISPR/Cas9 technology and high throughput screening.In this study,we obtained in situ knockin(KI)strains of all 20 Argonaute proteins with fusion tags by CRISPR/Cas9 technology in C.elegans,and one of them,wago-5,appeared to be another pseudogene.RNA immunoprecipitation against 19 endogenously expressed Argonautes and high-throughput sequencing,followed by bioinformatics analysis of the sRNAs enriched by Argonautes,acquired the sRNA profiles of individual Argonautes.In this study,we found that there were 10 Argonautes enriched miRNAs,17 Argonautes bound 22G-RNAs,8 Argonautes bound 26G-RNAs,and 1 Argonaute,PRG-1,bound piRNAs.Except for piRNAs,the other types of sRNAs could be bound by different Argonaute proteins,and all Argonautes,including PRG-1,can regulate different types of sRNAs,forming a complicated system composed of Argonautes and their regulatory sRNAs.Furthermore,Argonaute proteins regulate thousands of mRNAs,long non-coding RNAs(lncRNAs),pseudogenes,and transposons via associated 22G-RNAs and 26G-RNAs.The following GO analysis showed that 22G-RNAs or 26G-RNAs targeted mRNAs involved in many important biological processes in C.elegans.Intriguingly,there are also bidirectional,unidirectional,and self-regulating networks within Argonautes transcripts.Besides,we notice four Argonaute proteins,HRDE-1,WAGO-4,CSR-1,and PPW-2,that bind uridylated 22G-RNAs to varying degrees,and these Argonaute proteins play a certain role in mediating transgenerational inheritance induced by exogenous RNA interference.In addition,bioinformatics analysis also showed that the Argonaute proteins in C.elegans could bind sRNAs from E.coli,and sRNAs were proven to stabilize T08E11.1 with the aid of the C.elegans Argonaute protein,PPW-1.In summary,we portrayed the sRNAs bound to each functional Argonaute in C.elegans.Bioinformatics analyses combined with experimental investigations provided perceptions in the overall view of the regulatory network formed by C.elegans Argonautes and sRNAs.The generated in situ knockin(KI)strains and sRNA profiles bound to individual Argonautes will be valuable resources for further studies.