SUMMARY Enhancers control the correct temporal and cell type-specific activation of gene expression in higher eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. We use the FANTOM5 panel of samples covering the majority of human tissues and cell types to produce an atlas of active, in vivo transcribed enhancers. We show that enhancers share properties with CpG-poor mRNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, identify disease-associated regulatory single nucleotide polymorphisms, and classify cell type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell type-specific enhancers and gene regulation.

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we disclose a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA), and then further connects together with the ZC3H18 protein to the nuclear exosome targeting (NEXT) complex, forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5′cap links transcription termination to exosomal RNA degradation via CBCN.

Ntini et al. 3The non-protein-coding part of the human genome is pervasively transcribed into a large diversity of non-coding (nc) RNA 1 . A substantial fraction of this material derives from, or near, active gene promoters, that are producing a range of small-( 1-6 ) and long non-coding RNA (lncRNA) 7 . Indeed, it has been estimated that >60% of lncRNAs in human embryonic stem cells derive from promoters of active proteincoding genes 8 . Although some lncRNAs have reported functions, these species are generally kept at low abundance by cellular degradation activities 9,10 . For example, we previously coupled depletion of the major nuclear 3'-5' exonucleolytic activity, the RNA exosome, with tiling microarrays to reveal PROMPTs closely upstream of active human gene promoters 9 . PROMPTs are 5'capped, >100nt long and 3'end adenylated in the absence of exosome activity 11 . The mechanism underlying the efficient exosome-mediated suppression of these lncRNAs, while preserving the promoter-downstream mRNA, remains enigmatic.Here, we couple exosome-depletion to high-throughput 5'end-, 3'end-and regular RNA-sequencing (RNAseq) to create a genome-wide map of PROMPTs. Our results demonstrate that PROMPT transcription initiates antisense with respect to the downstream gene. We suggest that such initiating RNAPII, if stalled at a PROMPT-TSS proximal position, can elicit the production of previously reported TSSa-RNA.Sequence motifs around PROMPT 3'ends adhere to a pA site consensus and are significantly more abundant upstream than downstream of gene promoters. This provides a directional RNA output from human promoters by rapidly terminating antisense transcription and enforcing degradation of its RNA product. RESULTS PROMPTs initiate from bi-directional promoter activityTo obtain strand-specific and positional information of PROMPTs, we first subjected total RNA from HeLa cells, that had been treated with either a control (ctrl) eGFP siRNA or RRP40 siRNA ( Supplementary Fig. 1a), to regular RNA sequencing (RNAseq) as well as cap-selected RNA 5'end sequencing (Cap Analysis of Gene Expression (CAGE)). We focused our analysis on protein-coding genes and therefore considered reads mapping to the -3kb to +1kb regions of 2428 UCSC gene promoters, which were selected not to overlap any other annotated mRNAs. When aligned to the TSSs of these promoters, both RNAseq-and CAGE-data disclosed a strong presence of exosome-sensitive transcripts originating closely upstream of the gene TSS Ntini et al. 4(average peak CAGE position at -110bp) and commencing in the antisense direction relative to the neighboring mRNA TSS (Fig. 1a bottom panel, compare 'ctrl' and 'RRP40' plots). Only minor signal was detected in the sense direction of the same region. Whereas the abundance of antisense PROMPT (asPROMPT) CAGE tags increased by an average of 8-fold upon RRP40 depletion, the corresponding sense CAGE signals of the same region were largely unaffected (Fig. 1b, P<2e-16, twosided t-test). This predominant occurrence of asPROMPTs was also visi...

Splicing efficiency varies among transcripts, and tight control of splicing kinetics is crucial for coordinated gene expression. N-6-methyladenosine (m6A) is the most abundant RNA modification and is involved in regulation of RNA biogenesis and function. The impact of m6A on regulation of RNA splicing kinetics is unknown. Here, we provide a time-resolved high-resolution assessment of m6A on nascent RNA transcripts and unveil its importance for the control of RNA splicing kinetics. We find that early co-transcriptional m6A deposition near splice junctions promotes fast splicing, while m6A modifications in introns are associated with long, slowly processed introns and alternative splicing events. In conclusion, we show that early m6A deposition specifies the fate of transcripts regarding splicing kinetics and alternative splicing.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans.