New types of small RNAs distinct from microRNAs (miRNAs) are progressively being discovered in various organisms. In order to discover such novel small RNAs, a library of 17-to 26-base-long RNAs was created from prostate cancer cell lines and sequenced by ultra-high-throughput sequencing. A significant number of the sequences are derived from precise processing at the 59 or 39 end of mature or precursor tRNAs to form three series of tRFs (tRNA-derived RNA fragments): the tRF-5, tRF-3, and tRF-1 series. These sequences constitute a class of short RNAs that are second most abundant to miRNAs. Northern hybridization, quantitative RT-PCR, and splinted ligation assays independently measured the levels of at least 17 tRFs. To demonstrate the biological importance of tRFs, we further investigated tRF-1001, derived from the 39 end of a Ser-TGA tRNA precursor transcript that is not retained in the mature tRNA. tRF-1001 is expressed highly in a wide range of cancer cell lines but much less in tissues, and its expression in cell lines was tightly correlated with cell proliferation. siRNAmediated knockdown of tRF-1001 impaired cell proliferation with the specific accumulation of cells in G2, phenotypes that were reversed specifically by cointroducing a synthetic 29-O-methyl tRF-1001 oligoribonucleotide resistant to the siRNA. tRF-1001 is generated in the cytoplasm by tRNA 39-endonuclease ELAC2, a prostate cancer susceptibility gene. Our data suggest that tRFs are not random by-products of tRNA degradation or biogenesis, but an abundant and novel class of short RNAs with precise sequence structure that have specific expression patterns and specific biological roles.[Keywords: Small RNA; tRNA; deep sequencing; cancer cell proliferation] Supplemental material is available at http://www.genesdev.org.

We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200–400 bp long, derived from unique non-repetitive sequence and are enriched in the 5' untranslated regions of genes, exons and CpG islands. Chromosomal loci that are enriched sources of microDNA in adult brain are somatically mosaic for micro-deletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a new DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci. Single nucleotide polymorphisms and copy number variations are known sources of genetic variation between individuals (1–5), but there is also great interest in variations that arise during generation of somatic tissues like the mammalian brain, leading to genetic mosaicism between somatic cells. To identify sites of intramolecular homologous recombination during brain development, we searched for extrachromosomal circular DNA (eccDNA) derived from excised chromosomal regions in normal mouse embryonic brains.

Cell-free circulating linear DNA is being explored for non-invasive diagnosis and management of tumors and fetuses, the so-called liquid biopsy. Previously, we observed the presence of small extrachromosomal circular DNA (eccDNA), called microDNA, in the nuclei of mammalian tissues and cell lines. Now, we demonstrate that cell-free microDNA derived from uniquely mapping regions of the genome is detectable in plasma and serum from both mice and humans and that they are significantly longer (30–60% >250 bases) than cell-free circulating linear DNA (~150 bases). Tumor-derived human microDNA is detected in the mouse circulation in a mouse xenograft model of human ovarian cancer. Comparing the microDNA from paired tumor and normal lung tissue specimens reveals that the tumors contain longer microDNA. Consistent with human cancers releasing microDNA into the circulation, serum and plasma samples (12 lung and 11 ovarian cancer) collected prior to surgery are enriched for longer cell free microDNA compared to samples from the same patient obtained several weeks after surgical resection of the tumor. Thus, circular DNA in the circulation is a previously unexplored pool of nucleic acids that could complement microRNAs (miRs) and linear DNA for diagnosis and for intercellular communication.

SUMMARY MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density, from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveal that homologous recombination and nonhomologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology; either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.

Interest in extrachromosomal circular DNA (eccDNA) molecules has increased recently because of their widespread presence in normal cells across every species ranging from yeast to humans, their increased levels in cancer cells and their overlap with oncogenic and drug-resistant genes. However, the majority of eccDNA (microDNA) in mammalian tissues and cell lines are too small to carry protein coding genes. We have tested functional capabilities of microDNA by creating artificial microDNA molecules mimicking known microDNA sequences and have discovered that they express functional small regulatory RNA including microRNA and novel si-like RNA. MicroDNA are transcribed in vitro and in vivo independent of a canonical promoter sequence. MicroDNA that carry miRNA genes form transcripts that are processed by the endogenous RNA-interference pathway into mature miRNA molecules, which repress a luciferase reporter gene as well as endogenous mRNA targets of the miRNA. Further, microDNA that contain sequences of exons repress the endogenous gene from which the microDNA were derived through the formation of novel si-like RNA. We also show that endogenous microDNA associate with RNA polymerases subunits, POLR2H and POLR3F. Together, these results suggest that microDNA may modulate gene expression through the production of both known and novel regulatory small RNA.