Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained noncolinear exons (a "backsplice") and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.

The canonical histone proteins are encoded by replication-dependent genes and must rapidly reach high levels of expression during S phase. In metazoans the genes that encode these proteins produce mRNAs that, instead of being polyadenylated, contain a unique 3' end structure. By contrast, the synthesis of the variant, replication-independent histones, which are encoded by polyadenylated mRNAs, persists outside of S phase. Accurate positioning of both histone types in chromatin is essential for proper transcriptional regulation, the demarcation of heterochromatic boundaries and the epigenetic inheritance of gene expression patterns. Recent results suggest that the coordinated synthesis of replication-dependent and variant histone mRNAs is achieved by signals that affect formation of the 3' end of the replication-dependent histone mRNAs.Histones are the primary protein component of chromatin. Although they were initially thought to be mainly involved in chromosomal DNA packaging in eukaryotes, it is now recognized that they also have a crucial role in regulating gene expression. Histones can be extensively modified after translation and these modifications play an important part in regulating gene expression. They are constantly being shifted, modified, evicted and re-deposited as chromatin is continually remodelled (reviewed in REF. 1 ). Thus, the cell must carefully coordinate the replication of DNA, the synthesis of an estimated 10 8 molecules of each histone type in mammalian cells and the rapid deposition of new and old histones to reform chromatin during each relatively short S phase 2,3 .In metazoans the bulk of the histone proteins, defined here as the canonical histone proteins, are encoded by a family of replication-dependent histone genes. Their mRNAs are the only known cellular non-polyadenylated mRNAs in eukaryotes 4 . These genes encode all four core histones -H2A, H2B, H3 and H4 -which make up the nucleosome, and the linker H1 histones, which are found between nucleosomes. In place of a poly(A) tail, replicationdependent histone mRNAs end in a 3′ stem-loop sequence that is crucial in their regulation (FIG. 1a), and is formed by endonu-cleolytic cleavage of the pre-mRNA (FIG. 1b). This novel 3′ end results in the requirement for a distinct set of factors for metabolism and regulation of these histone mRNAs. These mRNAs must be expressed rapidly at the beginning of S phase and must persist at high levels throughout S phase to coincide with the replication of DNA. They are destroyed at the conclusion of S phase or rapidly during S phase if DNA replication is halted.In addition to the canonical histones, there are several variant histones whose synthesis is not cell-cycle-regulated and whose mRNAs are polyadenylated and expressed throughout the cell cycle (replication-independent histone mRNAs). These histones include the H3.3 and Correspondence to W.F.M.

Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain-leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.