Extrachromosomal circular DNA produces regulatory small RNAs involved in zygotic genome remodeling

Extrachromosomal circular DNA (eccDNA) is widely distributed in eukaryotes.ParameciumeccDNA is generated through random concatenation of short, transposon-derived sequences that are excised from the germline genomeand transcribed to generateregulatory small RNA.These small RNAs help to eliminate a bulk of transposon remnants from the zygotic somatic genome through a positive feedback loop mechanism. Here we present evidence that Paramecium eccDNA transcription depends oneukaryotic DNA-dependent RNA polymerase III (RNAP III) and knockdown of RNAP III subunit RPC2 results in specific loss of small RNA deriving from eccDNAand an incomplete elimination of transposable elements from the genome.We identified thousands of unique eccDNA sequences, each falling within the size range of 100 -600 bp. Furthermore, our research indicates that transcription start sites are distributed across the entire eccDNA sequences. This suggests that factors other than conserved sequence elements play a role in recruiting RNAP III to these unconventional DNA templates.