The Transcription Mechanism Of PiRNA In Caenorhabditis Elegans

Small RNAs exist in various organisms,such as humans,mice,flies and worms,and those small RNAs are both important and conservative.Among those small RNAs,piRNAs(PIWI-interacting RNAs)associate with PIWI proteins and play crucial roles in maintaining genome stability,silencing non-self sequences and maintaining male reproduction.In Caenorhabditis elegans,piRNAs are also referred to as 21U-RNAs because they have a length of 21 nucleotides with a uridine at their 5' end.piRNA genes in C.elegans are predominantly located on two piRNA clusters on chromosome IV.Transcription of piRNA genes relies on the upstream sequence transcription complex(USTC)composed of TOFU-5,TOFU-4,PRDE-1 and SNPC-4.After being transcribed by RNA polymerase II,piRNA precursors undergo a series of trimming and processing to produce mature piRNAs.However,how the USTC complex is recruited by piRNA genes to initiate piRNA transcription and the molecular mechanisms of piRNA transcription remain elusive.Here,we used classic genetic screen methods,molecular biology and cellular biology approaches to search for novel factors that are required for piRNA transcription in C.elegans.By using the USTC complex as a tool to conduct a forward genetic screen and identified a chromodomain-containing protein UAD-2 that was required for piRNA focus formation.We performed deep sequencing of total small RNAs from uad-2 mutants and confirmed that UAD-2 is involved in piRNA biogenesis.We constructed GFP::3xFLAG-tagged UAD-2 transgenes using CRISPR/Cas9 directed in situ gene editing technology.UAD-2::GFP exhibited distinct foci in the germline nuclei and colocalized with the USTC complex.In addition,piRNA focus formation of UAD-2 and the USTC complex was codependent in germline nuclei.We further used TOFU5::GFP transgene to perform a candidate-based reverse genetic screen and found that the PRC2 complex,two chromatin remodeling factors ISW-1 and MRG-1,SUN-1 and R151.8 were required for piRNA focus formation of the USTC complex and UAD-2 in the germline nuclei.By performing chromatin immunoprecipitation assays,piRNA sensor and piRNA deep sequencing assays,we found that the PRC2 complex,ISW-1 and MRG-1 were required for the USTC complex binding to piRNA genes and piRNA biogenesis.Besides that,we found that both the USTC complex and UAD-2 failed to aggregate at piRNA foci in the germline nuclei when animals were grown at 25?,reducing the level of two types of piRNAs.Via pull down assay in vitro,we confirmed that the chromodomain of UAD-2 bound H3K27me3 peptides through pull down in vitro.In summary,we found a novel chromodomain-containing protein UAD-2 and revealed functions of UAD-2,the PRC2 complex and two chromatin remodeling proteins ISW-1 and MRG-1 in piRNA production.We further confirmed that UAD-2 bound H3K27me3 via its chromodomain.This study will provide new insights into understanding the mechanism of piRNA biogenesis in C.elegans.