Impaired lipid metabolism in astrocytes underlies degeneration of cortical projection neurons in hereditary spastic paraplegia.

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.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. 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.

Red blood cell distribution width: Genetic evidence for aging pathways in 116,666 volunteers.

INTRODUCTION: 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. RESULTS: 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. CONCLUSIONS: 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.

Incidence of pancreatic cancer is dramatically increased by a high fat, high calorie diet in KrasG12D mice.

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.

Global Analysis of Mouse Polyomavirus Infection Reveals Dynamic Regulation of Viral and Host Gene Expression and Promiscuous Viral RNA Editing.

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.

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila.

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified "high occupancy target" (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteins and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. From the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.

Regulation of alternative splicing in Drosophila by 56 RNA binding proteins.

Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

Genome-wide identification of zero nucleotide recursive splicing in Drosophila.

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. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing. 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.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.

Diversity and dynamics of the Drosophila transcriptome.

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.

Global patterns of tissue-specific alternative polyadenylation in Drosophila.

We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3' UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3' UTR length in the nervous system.

Genomewide characterization of non-polyadenylated RNAs.

BACKGROUND: RNAs can be physically classified into poly(A)+ or poly(A)- transcripts according to the presence or absence of a poly(A) tail at their 3' ends. Current deep sequencing approaches largely depend on the enrichment of transcripts with a poly(A) tail, and therefore offer little insight into the nature and expression of transcripts that lack poly(A) tails. RESULTS: We have used deep sequencing to explore the repertoire of both poly(A)+ and poly(A)- RNAs from HeLa cells and H9 human embryonic stem cells (hESCs). Using stringent criteria, we found that while the majority of transcripts are poly(A)+, a significant portion of transcripts are either poly(A)- or bimorphic, being found in both the poly(A)+ and poly(A)- populations. Further analyses revealed that many mRNAs may not contain classical long poly(A) tails and such messages are overrepresented in specific functional categories. In addition, we surprisingly found that a few excised introns accumulate in cells and thus constitute a new class of non-polyadenylated long non-coding RNAs. Finally, we have identified a specific subset of poly(A)- histone mRNAs, including two histone H1 variants, that are expressed in undifferentiated hESCs and are rapidly diminished upon differentiation; further, these same histone genes are induced upon reprogramming of fibroblasts to induced pluripotent stem cells. CONCLUSIONS: We offer a rich source of data that allows a deeper exploration of the poly(A)- landscape of the eukaryotic transcriptome. The approach we present here also applies to the analysis of the poly(A)- transcriptomes of other organisms.

Conservation of an RNA regulatory map between Drosophila and mammals.

Alternative splicing is generally controlled by proteins that bind directly to regulatory sequence elements and either activate or repress splicing of adjacent splice sites in a target pre-mRNA. Here, we have combined RNAi and mRNA-seq to identify exons that are regulated by Pasilla (PS), the Drosophila melanogaster ortholog of mammalian NOVA1 and NOVA2. We identified 405 splicing events in 323 genes that are significantly affected upon depletion of ps, many of which were annotated as being constitutively spliced. The sequence regions upstream and within PS-repressed exons and downstream from PS-activated exons are enriched for YCAY repeats, and these are consistent with the location of these motifs near NOVA-regulated exons in mammals. Thus, the RNA regulatory map of PS and NOVA1/2 is highly conserved between insects and mammals despite the fact that the target gene orthologs regulated by PS and NOVA1/2 are almost entirely nonoverlapping. This observation suggests that the regulatory codes of individual RNA binding proteins may be nearly immutable, yet the regulatory modules controlled by these proteins are highly evolvable.

The transcriptional diversity of 25 Drosophila cell lines.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.

The developmental transcriptome of Drosophila melanogaster.

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.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Global analysis of trans-splicing in Drosophila.

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 trans-splicing 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.

Regulatory divergence in Drosophila revealed by mRNA-seq.

The regulation of gene expression is critical for organismal function and is an important source of phenotypic diversity between species. Understanding the genetic and molecular mechanisms responsible for regulatory divergence is therefore expected to provide insight into evolutionary change. Using deep sequencing, we quantified total and allele-specific mRNA expression levels genome-wide in two closely related Drosophila species (D. melanogaster and D. sechellia) and their F(1) hybrids. We show that 78% of expressed genes have divergent expression between species, and that cis- and trans-regulatory divergence affects 51% and 66% of expressed genes, respectively, with 35% of genes showing evidence of both. This is a relatively larger contribution of trans-regulatory divergence than was expected based on prior studies, and may result from the unique demographic history of D. sechellia. Genes with antagonistic cis- and trans-regulatory changes were more likely to be misexpressed in hybrids, consistent with the idea that such regulatory changes contribute to hybrid incompatibilities. In addition, cis-regulatory differences contributed more to divergent expression of genes that showed additive rather than nonadditive inheritance. A correlation between sequence similarity and the conservation of cis-regulatory activity was also observed that appears to be a general feature of regulatory evolution. Finally, we examined regulatory divergence that may have contributed to the evolution of a specific trait--divergent feeding behavior in D. sechellia. Overall, this study illustrates the power of mRNA sequencing for investigating regulatory evolution, provides novel insight into the evolution of gene expression in Drosophila, and reveals general trends that are likely to extend to other species.

RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.

Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.