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Many proteins regulate the expression of genes by binding to specific regions encoded in the genome 1 . Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs. A combination of five assays is used to produce a catalogue of RNA elements to which RNA-binding proteins bind in human cells.

Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.

In flies, some introns contain internal splice sites that cause ‘recursive splicing’, a multi-step removal of a single intron; this study demonstrates that the scope of this regulatory mechanism is much more extensive in flies than had been appreciated, and provides details about the recursive splicing process. Recursive splicing in insects and vertebrates The mechanisms by which the very longest genes in eukaryotic genomes are accurately processed are poorly understood. It was thought that intron removal generally involved a single excisive step. Later studies showed that, in flies, some introns contain internal splice sites that cause 'recursive splicing', in which single introns are removed 'bit-by-bit' in several sequential splicing reactions. Brenton Graveley and coworkers demonstrate that the scope of this regulatory mechanism is much more extensive in flies than had been appreciated. They identify nearly 200 zero-nucleotide exons in Drosophila that are the products of recursive splicing. Jernej Ule and colleagues identify recursive splicing sites in vertebrates, particularly within long genes encoding proteins that are involved in neuronal development. Analysis of the mechanism of their splicing reveals that such splicing sites can be used to dictate different mRNA isoforms. Recursive splicing is a process in which large introns are removed in multiple steps by re-splicing at ratchet points—5′ splice sites recreated after splicing 1 . Recursive splicing was first identified in the Drosophila Ultrabithorax ( Ubx ) gene 1 and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing 2 , 3 . Here we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues and cultured cells. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, that depletion of U2AF inhibits recursive splicing, and that the sequence and function of ratchet points are evolutionarily conserved in Drosophila . Finally, we identify four recursively spliced human genes, one of which is also recursively spliced in Drosophila . Together, these results indicate that recursive splicing is commonly used in Drosophila , occurs in humans, and provides insight into the mechanisms by which some large introns are removed.

Variability in red blood cell volumes (distribution width, RDW) increases with age and is strongly predictive of mortality, incident coronary heart disease and cancer. We investigated inherited genetic variation associated with RDW in 116,666 UK Biobank human volunteers. A large proportion RDW is explained by genetic variants (29%), especially in the older group (60+ year olds, 33.8%, <50 year olds, 28.4%). RDW was associated with 194 independent genetic signals; 71 are known for conditions including autoimmune disease, certain cancers, BMI, Alzheimer's disease, longevity, age at menopause, bone density, myositis, Parkinson's disease, and age-related macular degeneration. Exclusion of anemic participants did not affect the overall findings. Pathways analysis showed enrichment for telomere maintenance, ribosomal RNA, and apoptosis. The majority of RDW-associated signals were intronic (119 of 194), including SNP rs6602909 located in an intron of oncogene GAS6, an eQTL in whole blood. Although increased RDW is predictive of cardiovascular outcomes, this was not explained by known CVD or related lipid genetic risks, and a RDW genetic score was not predictive of incident disease. The predictive value of RDW for a range of negative health outcomes may in part be due to variants influencing fundamental pathways of aging.

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A) + RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites. A large-scale transcriptome analysis in Drosophila melanogaster , across tissues, cell types and conditions, provides insights into global patterns and diversity of transcription initiation, splicing, polyadenylation and non-coding RNA expression.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development. Elements of gene function Three papers in this issue of Nature report on the modENCODE initiative, which aims to characterize functional DNA elements in the fruitfly Drosophila melanogaster and the roundworm Caenorhabditis elegans . Kharchenko et al . present a genome-wide chromatin landscape of the fruitfly, based on 18 histone modifications. They describe nine prevalent chromatin states. Integrating these analyses with other data types reveals individual characteristics of different genomic elements. Graveley et al . have used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages of the fruitfly. Among the results are scores of new genes, coding and non-coding transcripts, as well as splicing and editing events. Finally, Nègre et al . have produced a map of the regulatory part of the fruitfly genome, defining a vast array of putative regulatory elements, such as enhancers, promoters, insulators and silencers. As part of the modENCODE initiative, which aims to characterize functional DNA elements in D. melanogaster and C. elegans , this study uses RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages of the fruitfly. Among the results are scores of new genes, coding and non-coding transcripts, as well as splicing and editing events.

Mouse polyomavirus (MPyV) lytically infects mouse cells, transforms rat cells in culture, and is highly oncogenic in rodents. We have used deep sequencing to follow MPyV infection of mouse NIH3T6 cells at various times after infection and analyzed both the viral and cellular transcriptomes. Alignment of sequencing reads to the viral genome illustrated the transcriptional profile of the early-to-late switch with both early-strand and late-strand RNAs being transcribed at all time points. A number of novel insights into viral gene expression emerged from these studies, including the demonstration of widespread RNA editing of viral transcripts at late times in infection. By late times in infection, 359 host genes were seen to be significantly upregulated and 857 were downregulated. Gene ontology analysis indicated transcripts involved in translation, metabolism, RNA processing, DNA methylation, and protein turnover were upregulated while transcripts involved in extracellular adhesion, cytoskeleton, zinc finger binding, SH3 domain, and GTPase activation were downregulated. The levels of a number of long noncoding RNAs were also altered. The long noncoding RNA MALAT1, which is involved in splicing speckles and used as a marker in many late-stage cancers, was noticeably downregulated, while several other abundant noncoding RNAs were strongly upregulated. We discuss these results in light of what is currently known about the MPyV life cycle and its effects on host cell growth and metabolism.

Precursor mRNA (pre-mRNA) splicing can join exons contained on either a single pre-mRNA (cis) or on separate pre-mRNAs (trans). It is exceedingly rare to have trans-splicing between protein-coding exons and has been demonstrated for only two Drosophila genes: mod(mdg4) and lola. It has also been suggested that trans-splicing is a mechanism for the generation of chimeric RNA products containing sequence from multiple distant genomic sites. Because most high-throughput approaches cannot distinguish cis- and transsplicing events, the extent to which trans-splicing occurs between protein-coding exons in any organism is unknown. Here, we used paired-end deep sequencing of mRNA to identify genes that undergo trans-splicing in Drosophila interspecies hybrids. We did not observe credible evidence for the existence of chimeric RNAs generated by trans-splicing of RNAs transcribed from distant genomic loci. Rather, our data suggest that experimental artifacts are the source of most, if not all, apparent chimeric RNA products. We did, however, identify 80 genes that appear to undergo trans-splicing between homologous alleles and can be classified into three categories based on their organization: (i) genes with multiple 3' terminal exons, (ii) genes with multiple first exons, and (iii) genes with very large introns, often containing other genes. Our results suggest that trans-splicing between homologous alleles occurs more commonly in Drosophila than previously believed and may facilitate expression of architecturally complex genes.

Hereditary spastic paraplegias (HSPs) are caused by a length-dependent axonopathy of long corticospinal neurons, but how axons of these cortical projection neurons (PNs) degenerate remains elusive. We generated isogenic human pluripotent stem cell (hPSC) lines for two ATL1 missense mutations associated with SPG3A, the most common early-onset autosomal dominant HSP. In hPSC-derived cortical PNs, ATL1 mutations resulted in reduced axonal outgrowth, impaired axonal transport, and accumulated axonal swellings, recapitulating disease-specific phenotypes. Importantly, ATL1 mutations dysregulated proteolipid gene expression, reduced lipid droplet size in astrocytes, and unexpectedly disrupted cholesterol transfer from glia to neurons, leading to cholesterol deficiency in SPG3A cortical PNs. Applying cholesterol or conditioned medium from control astrocytes, a major source of cholesterol in the brain, rescued aberrant axonal transport and swellings in SPG3A cortical PNs. Furthermore, treatment with the NR1H2 agonist GW3965 corrected lipid droplet defects in SPG3A astrocytes and promoted cholesterol efflux from astrocytes, leading to restoration of cholesterol levels and rescue of axonal degeneration in SPG3A cortical PNs. These results reveal a non-cell autonomous mechanism underlying axonal degeneration of cortical PNs mediated by impaired cholesterol homeostasis in glia.

Histone deacetylases (HDACs) are important cancer drug targets. Existing FDA-approved drugs target the catalytic pocket of HDACs, which is conserved across subfamilies (classes) of HDAC. However, engineering specificity is an important goal. Herein, we use molecular modeling approaches to identify and target potential novel pockets specific to Class IIA HDAC-HDAC4 at the interface between HDAC4 and the transcriptional corepressor component protein NCoR. These pockets were screened using an ensemble docking approach combined with consensus scoring to identify compounds with a different binding mechanism than the currently known HDAC modulators. Binding was compared in experimental assays between HDAC4 and HDAC3, which belong to a different family of HDACs. HDAC4 was significantly inhibited by compound 88402 but not HDAC3. Two other compounds (67436 and 134199) had IC50 values in the low micromolar range for both HDACs, which is comparable to the known inhibitor of HDAC4, SAHA (Vorinostat). However, both of these compounds were significantly weaker inhibitors of HDAC3 than SAHA and thus more selective, albeit to a limited extent. Five compounds exhibited activity on human breast carcinoma and/or urothelial carcinoma cell lines. The present result suggests potential mechanistic and chemical approaches for developing selective HDAC4 modulators.