Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms.

Comparative RNA-seq analysis of two related pathogenic and non-pathogenic bacterial strains reveals a hidden layer of divergence in the non-coding genome as well as conserved, widespread regulatory structures called ‘Excludons', which mediate regulation through long non-coding antisense RNAs.

N6-methyladenosine (m 6 A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m 6 A stoichiometry and the lack of antibodyindependent methodologies for interrogating m 6 A.Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m 6 A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m 6 A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is ''hard coded'' in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m 6 A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m 6 A regulation in subcellular fractions, diverse cell types, and disease states. (A) Distribution of cleavage efficiencies (y axis) in RNA extracted from WT ime4D/D strains with versus without m 6 A-IP treatment. (B) Clustered pairwise correlation of the samples in (A). (C) Empirical false-detection rates per confidence group. (D) Distribution of m 6 A-seq sites across the confidence groups defined via MAZTER-seq. (E) Distribution of m 6 A-seq scores from Schwartz et al. (2013) by MAZTER-seq confidence groups. (F) Sequence logos for sites identified via MAZTER-seq shown separately for the indicated confidence groups. (G) Higher-confidence sites are closer to the end of the transcript. Distributions of 3 0 end distances by confidence group. (H) SCARLET-based readouts of methylation levels at each of the indicated sites.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

Transcription of a gene usually ends at a regulated termination point, preventing the RNA-polymerase from reading through the next gene. However, sporadic reports suggest that chimeric transcripts, formed by transcription of two consecutive genes into one RNA, can occur in human. The splicing and translation of such RNAs can lead to a new, fused protein, having domains from both original proteins. Here, we systematically identified over 200 cases of intergenic splicing in the human genome (involving 421 genes), and experimentally demonstrated that at least half of these fusions exist in human tissues. We showed that unique splicing patterns dominate the functional and regulatory nature of the resulting transcripts, and found intergenic distance bias in fused compared with nonfused genes. We demonstrate that the hundreds of fused genes we identified are only a subset of the actual number of fused genes in human. We describe a novel evolutionary mechanism where transcription-induced chimerism followed by retroposition results in a new, active fused gene. Finally, we provide evidence that transcription-induced chimerism can be a mechanism contributing to the evolution of protein complexes.[Supplemental material is available online at www.genome.org.]Eukaryotic genes are generally well defined on the genome. Transcription usually begins from a transcription start site, which is guided by the promoter, and ends at a regulated termination point (Zhao et al. 1999;Proudfoot et al. 2002). Consecutive genes are usually separated from each other by intergenic, nonexpressed regions (Lander et al. 2001). In recent years, however, evidence for the existence of mammalian transcripts that span two adjacent, independent genes, have emerged. Typically, such chimeric transcripts begin at the promoter of the upstream gene and end at the termination point of the downstream gene. The intergenic region is spliced out of the transcript as an intron, so that the resulting fused transcripts possess exons from the two different genes (Fig. 1). This phenomenon was coined intergenic splicing or cotranscription and considered extremely rare. In human, only a handful of such transcription-induced chimeras (TICs; see also the accompanying paper by Parra et al. 2006 in this issue) were so far reported (Table 1).Fused RNAs were shown to be regulated and to have unique expression patterns. For example, the HHLA1-OC90 fusion transcript is restricted to teratocarcinoma cell lines while absent from normal cells (Kowalski et al. 1999). In the case of LY75-CD302 (CD205-DCL1) fusion, the chimera is predominant in Hodgkin and Reed-Strenberg cell lines (Kato et al. 2003). The fusion can change the properties of the participating proteins, or change their localization, such as in the case of Kua-UBE2Vl fusion, where the fused protein is localized to the cytoplasm, while UBE2Vl is a nuclear protein (Thomson et al. 2000). The most characterized human fusion transcript is of two members of the TNF ligand family, TNFSF12 (previously known as TWEAK) and TNFSF1...