The Upstream Sequence Transcription Complex(USTC) Regulate PiRNA Transcription

Small RNAs are significant molecules that can regulate gene expression.Since the discovery of RNA interfenrence,classes of small RNAs have been discovered s in Caenorhabditis elegans.Among these small RNAs,piRNA is highly conserved.piRNAs suppress transposons,nonself nucleic acids,maintain genome integrity and are essential for fertility in a variety of organisms.In Caenorhabditis elegans,piRNA initiate transgenerational gene silence.Most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units.Later,piRNA transcripts are processed before engaging Piwi protein.While a few genes have been shown to be required for piRNA biogenesis,the mechanism of piRNA transcription and process remains elusive.Here,we used genetics,cell biology and molecular biology approaches to study piRNA biogenesis mechanism in Caenorhabditis elegans.Fisrtly,we generated single-copy GFP-3xFlag-tagged TOFUs(Twenty-One-u Fouled Ups)transgenic strains using the Mosl-mediated single-copy insertion(MosSCI)technology.TOFU-1,TOFU-2,and TOFU-7 are expressed in germline cytoplasm.TOFU-3,TOFU-4 and TOFU-5 are colocalization with PRDE-1 in germline nucleus.We found that PRDE-1,SNPC-4,TOFU-4 and the SANT domain of TOFU-5 are required for the subcellular location and chromatin binding of TOFU-5 by image and ChIP assay.PRDE-1,SNPC-4 and TOFU-5 are required for the subcellular location and chromatin binding of TOFU-4.By ChIP-seq,we found that PRDE-1,SNPC-4,TOFU-4 and TOFU-5 bind to the two piRNA clusters on chromosome IV and locate upstream piRNA genes.However,PRDE-1 and TOFU-4 only bind to piRNAs with Ruby motif upstream within piRNA clusters,SNPC-4 and TOFU-5 bind to all piRNAs and also other protein-coding or non-coding RNA genes.We identified an upstream sequence transcription complex(USTC)that is essential for piRNA biogenesis.The USTC contains PRDE-1,SNPC-4,TOFU-4 and TOFU-5.TOFU-6 is expressed in germline cytoplasm and P-granule engaged in piRNA processing.Finally,we found some novel factors associated with piRNA transcription or chromosome structure by TOFU-5::GFP foci.Using reverse genetic screen,we found new genes involved in TOFU-5::GFP foci formation including tbp-l,mes-2,mes-3 and mes-6,TOFU-5::GFP foci disappeared by feeding tbp-1,mes-2,mes-3 and mes-6 RNAi.In addition,we found an unknown gene pfd-1(piRNA foci defective 1)required for TOFU-5::GFP foci by forward genetic screen.Hence,we developed a forward genetic screen to identify new piRNA biogenesis and chromosome structure factors using TOFU-5::GFP reporter.