The PICS Complex Is Required For PiRNA Biogenesis And Chromosome Segregation

Small non-coding RNAs are not translated into proteins but regulate gene expression through complementary base pairing with target sequences.These small RNAs form the RNA-induced silencing complexes(RISCs)together with the Argonaute proteins.Among these Argonautes,piwi proteins are guided to specific targets by small guide RNA,referred to as-piwi-interacting RNAs or piRNAs.piRNAs play significant roles in suppressing transposons and non-self nucleic acids,maintaining genome integrity,and defending against viral infections.The biogenesis of piRNAs is not fully understood.In Caenorhabditis elegans,piRNA precursors are transcribed from thousands of genomic loci in germline nucleus and then transported and processed in the cytoplasm.In C.elegans,piRNAs are termed as 21U RNAs since most mature piRNAs are 21 nucleotides(nt)in length and start with 5'-monophosphorylated uracil.21U RNAs are loaded into piwi protein,PRG-1.Even though genetic screens have uncovered several 21U RNA biogenesis factors,little is known about how these factors interact or what they do.Here,we used genetics,molecular biology and cell biology approaches to study the piRNA biogenesis mechanisms based on previous identified 21U RNA biogenesis factors TOFUs(Twenty-One-u RNAs Fouled Ups).We generated GFP::3xflag-tagged TOFUs transgenic worms through MosSCI technology to probe the subcellular localization of these proteins.Among these transgenes,TOFU-3/4/5s are localized in nucleus and act in the transcription of piRNA while TOFU-1/2s are expressed in germline cytoplasm.Strikingly,TOFU-6 is present not only in the germline cytoplasm,but also at distinct peri-nuclear foci.Using immunoprecipitation and label-free quantitative mass spectrometry(IP-MS),We found several TOFU-6 interacting proteins.Then we assayed the protein-protein interactions of these proteins in vitro through GST-pull down and in vivo through IP-MS,and identified a piRNA biogenesis and chromosome segregation(PICS)complex.The PICS complex contains TOFU-6,PICS-1,ERH-2,PID-1 and TOST-1,which exhibit dynamic localization among different subcellular compartments.The PICS subunits,TOFU-6/PICS-1/ERH-2/PID-1,are largely concentrated at the germline perinuclear granule zone,and engage in piRNA processing,while the TOFU-6/PICS-1/ERH-2/TOST-1 subunits accumulate in embryos,and play essential roles in chromosome segregation.The functions of these factors in mediating chromosome segregation are independent of piRNA production.Our work described a key complex for 21U processing in C.and facilitates the understanding of 21U RNA pathway.