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Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. A study from the FANTOM consortium using single-molecule cDNA sequencing of transcription start sites and their usage in human and mouse primary cells, cell lines and tissues reveals insights into the specificity and diversity of transcription patterns across different mammalian cell types. Mapping the human transcription FANTOM5 (standing for functional annotation of the mammalian genome 5) is the fifth major stage of a major international collaboration that aims to dissect the transcriptional regulatory networks that define every human cell type. Two Articles in this issue of Nature present some of the project's latest results. The first paper uses the FANTOM5 panel of tissue and primary cell samples to define an atlas of active, in vivo bidirectionally transcribed enhancers across the human body. These authors show that bidirectional capped RNAs are a signature feature of active enhancers and identify more than 40,000 enhancer candidates from over 800 human cell and tissue samples. The enhancer atlas is used to compare regulatory programs between different cell types and identify disease-associated regulatory SNPs, and will be a resource for studies on cell-type-specific enhancers. In the second paper, single-molecule sequencing is used to map human and mouse transcription start sites and their usage in a panel of distinct human and mouse primary cells, cell lines and tissues to produce the most comprehensive mammalian gene expression atlas to date. The data provide a plethora of insights into open reading frames and promoters across different cell types in addition to valuable annotation of mammalian cell-type-specific transcriptomes.

Although the Human Genome Project was completed 4 years ago, the catalog of human protein-coding genes remains a matter of controversy. Current catalogs list a total of [almost equal to]24,500 putative protein-coding genes. It is broadly suspected that a large fraction of these entries are functionally meaningless ORFs present by chance in RNA transcripts, because they show no evidence of evolutionary conservation with mouse or dog. However, there is currently no scientific justification for excluding ORFs simply because they fail to show evolutionary conservation: the alternative hypothesis is that most of these ORFs are actually valid human genes that reflect gene innovation in the primate lineage or gene loss in the other lineages. Here, we reject this hypothesis by carefully analyzing the nonconserved ORFs--specifically, their properties in other primates. We show that the vast majority of these ORFs are random occurrences. The analysis yields, as a by-product, a major revision of the current human catalogs, cutting the number of protein-coding genes to [almost equal to]20,500. Specifically, it suggests that nonconserved ORFs should be added to the human gene catalog only if there is clear evidence of an encoded protein. It also provides a principled methodology for evaluating future proposed additions to the human gene catalog. Finally, the results indicate that there has been relatively little true innovation in mammalian protein-coding genes.

Background Glioblastoma multiform (GBM) is a devastating brain tumor with maximum surgical resection, radiotherapy plus concomitant and adjuvant temozolomide (TMZ) as the standard treatment. Diverse clinicopathological and molecular features are major obstacles to accurate predict survival and evaluate the efficacy of chemotherapy or radiotherapy. Reliable prognostic biomarkers are urgently needed for postoperative GBM patients. Methods The protein coding genes (PCGs) and long non-coding RNA (lncRNA) gene expression profiles of 233 GBM postoperative patients were obtained from The Cancer Genome Atlas (TCGA), TANRIC and Gene Expression Omnibus (GEO) database. We randomly divided the TCGA set into a training ( n  = 76) and a test set ( n  = 77) and used GSE7696 ( n  = 80) as an independent validation set. Survival analysis and the random survival forest algorithm were performed to screen survival associated signature. Results Six PCGs (EIF2AK3, EPRS, GALE, GUCY2C, MTHFD2, RNF212) and five lncRNAs (CTD-2140B24.6, LINC02015, AC068888.1, CERNA1, LINC00618) were screened out by a risk score model and formed a PCG-lncRNA signature for its predictive power was strongest (AUC = 0.78 in the training dataset). The PCG-lncRNA signature could divide patients into high- risk or low-risk group with significantly different survival (median 7.47 vs. 18.27 months, log-rank test P  < 0.001) in the training dataset. Similar result was observed in the test dataset (median 11.40 vs. 16.80 months, log-rank test P  = 0.001) and the independent set (median 8.93 vs. 16.22 months, log-rank test P  = 0.007). Multivariable Cox regression analysis verified that it was an independent prognostic factor for the postsurgical patients with GBM. Compared with IDH mutation status, O -(6)-methylguanine DNA methyltransferase promoter methylation status and age, the signature was proved to have a superior predictive power. And stratified analysis found that the signature could further separated postoperative GBM patients who received TMZ-chemoradiation into high- and low-risk groups in TCGA and GEO dataset. Conclusions The PCG-lncRNA signature was a novel prognostic marker to predict survival and TMZ-chemoradiation response in GBM patients after surgery.

Key Points Sexual dimorphism in gene expression is widespread across organisms and genomes. Genes with sex-biased expression, especially those with male-biased expression, tend to evolve rapidly in both protein sequence and expression level. Sex-biased genes are non-randomly distributed in the genome, with examples of both under-representation and over-representation on the X chromosome. There is mounting evidence that positive selection is the driving force behind the rapid evolution of sex-biased genes. This is probably caused by sexual selection and antagonistic coevolution between the sexes. Sex-linked genes that escape dosage compensation constitute a special case of sex-biased gene expression. There are several scenarios for the origin of sex-biased genes, including single-locus antagonism, sexual antagonism plus gene duplication and duplication of sex-biased genes. Most of the differences between males and females are due to differences in expression levels of certain genes. These genes have several interesting properties, such as rapid sequence evolution and an odd distribution across the genome. Differences between males and females in the optimal phenotype that is favoured by selection can be resolved by the evolution of differential gene expression in the two sexes. Microarray experiments have shown that such sex-biased gene expression is widespread across organisms and genomes. Sex-biased genes show unusually rapid sequence evolution, are often labile in their pattern of expression, and are non-randomly distributed in the genome. Here we discuss the characteristics and expression of sex-biased genes, and the selective forces that shape this previously unappreciated source of phenotypic diversity. Sex-biased gene expression has implications beyond just evolutionary biology, including for medical genetics.

The development of ribosomal profiling (Riboseq) revealed the immense coding capacity of human and viral genomes. Here, we used Riboseq to delineate the translatome of HIV-1 in infected CD4 + T cells. In addition to canonical viral protein coding sequences (CDSs), we identify 98 alternative open reading frames (ARFs), corresponding to small Open Reading Frames (sORFs) that are distributed across the HIV genome including the UTR regions. Using a database of HIV genomes, we observe that most ARF amino-acid sequences are likely conserved among clade B and C of HIV-1, with 8 ARF-encoded amino-acid sequences being more conserved than the overlapping CDSs. Using T cell-based assays and mass spectrometry-based immunopeptidomics, we demonstrate that ARFs encode viral polypeptides. In the blood of people living with HIV, ARF-derived peptides elicit potent poly-functional T cell responses mediated by both CD4 + and CD8 + T cells. Our discovery expands the list of conserved viral polypeptides that are targets for vaccination strategies and might reveal the existence of viral microproteins or pseudogenes. Here, using ribosomal profiling, the authors characterize the translatome of HIV-1 revealing tens of alternative open reading frames (ARF) that encode conserved viral antigens and show that ARF-derived peptides elicit potent HIV-specific poly-functional immune responses mediated by both CD4 + and CD8 +  T cells.

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1-encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.

Methylation of histone H3K36 in higher eukaryotes is mediated by multiple methyltransferases. Set2-related H3K36 methyltransferases are targeted to genes by association with RNA Polymerase II and are involved in preventing aberrant transcription initiation within the body of genes. The targeting and roles of the NSD family of mammalian H3K36 methyltransferases, known to be involved in human developmental disorders and oncogenesis, are not known. We used genome-wide chromatin immunoprecipitation (ChIP) to investigate the targeting and roles of the Caenorhabditis elegans NSD homolog MES-4, which is maternally provided to progeny and is required for the survival of nascent germ cells. ChIP analysis in early C. elegans embryos revealed that, consistent with immunostaining results, MES-4 binding sites are concentrated on the autosomes and the leftmost approximately 2% (300 kb) of the X chromosome. MES-4 overlies the coding regions of approximately 5,000 genes, with a modest elevation in the 5' regions of gene bodies. Although MES-4 is generally found over Pol II-bound genes, analysis of gene sets with different temporal-spatial patterns of expression revealed that Pol II association with genes is neither necessary nor sufficient to recruit MES-4. In early embryos, MES-4 associates with genes that were previously expressed in the maternal germ line, an interaction that does not require continued association of Pol II with those loci. Conversely, Pol II association with genes newly expressed in embryos does not lead to recruitment of MES-4 to those genes. These and other findings suggest that MES-4, and perhaps the related mammalian NSD proteins, provide an epigenetic function for H3K36 methylation that is novel and likely to be unrelated to ongoing transcription. We propose that MES-4 transmits the memory of gene expression in the parental germ line to offspring and that this memory role is critical for the PGCs to execute a proper germline program.

Lateral gene transfers (LGT), species to species transmission of genes by means other than direct inheritance from a common ancestor, have played significant role in shaping prokaryotic genomes and are involved in gain or transfer of important biological processes. Whether LGT significantly contributed to the composition of an animal genome is currently unclear. In nematodes, multiple LGT are suspected to have favored emergence of plant-parasitism. With the availability of whole genome sequences it is now possible to assess whether LGT have significantly contributed to the composition of an animal genome and to establish a comprehensive list of these events. We generated clusters of homologous genes and automated phylogenetic inference, to detect LGT in the genomes of root-knot nematodes and found that up to 3.34% of the genes originate from LGT of non-metazoan origin. After their acquisition, the majority of genes underwent series of duplications. Compared to the rest of the genes in these species, several predicted functional categories showed a skewed distribution in the set of genes acquired via LGT. Interestingly, functions related to metabolism, degradation or modification of carbohydrates or proteins were substantially more frequent. This suggests that genes involved in these processes, related to a parasitic lifestyle, have been more frequently fixed in these parasites after their acquisition. Genes from soil bacteria, including plant-pathogens were the most frequent closest relatives, suggesting donors were preferentially bacteria from the rhizosphere. Several of these bacterial genes are plasmid-borne, pointing to a possible role of these mobile genetic elements in the transfer mechanism. Our analysis provides the first comprehensive description of the ensemble of genes of non-metazoan origin in an animal genome. Besides being involved in important processes regarding plant-parasitism, genes acquired via LGT now constitute a substantial proportion of protein-coding genes in these nematode genomes.

Background Genome size and gene content in bacteria are associated with their lifestyles. Obligate intracellular bacteria (i.e., mutualists and parasites) have small genomes that derived from larger free-living bacterial ancestors; however, the different steps of bacterial specialization from free-living to intracellular lifestyle have not been studied comprehensively. The growing number of available sequenced genomes makes it possible to perform a statistical comparative analysis of 317 genomes from bacteria with different lifestyles. Results Compared to free-living bacteria, host-dependent bacteria exhibit fewer rRNA genes, more split rRNA operons and fewer transcriptional regulators, linked to slower growth rates. We found a function-dependent and non-random loss of the same 100 orthologous genes in all obligate intracellular bacteria. Thus, we showed that obligate intracellular bacteria from different phyla are converging according to their lifestyle. Their specialization is an irreversible phenomenon characterized by translation modification and massive gene loss, including the loss of transcriptional regulators. Although both mutualists and parasites converge by genome reduction, these obligate intracellular bacteria have lost distinct sets of genes in the context of their specific host associations: mutualists have significantly more genes that enable nutrient provisioning whereas parasites have genes that encode Types II, IV, and VI secretion pathways. Conclusion Our findings suggest that gene loss, rather than acquisition of virulence factors, has been a driving force in the adaptation of parasites to eukaryotic cells. This comparative genomic analysis helps to explore the strategies by which obligate intracellular genomes specialize to particular host-associations and contributes to advance our knowledge about the mechanisms of bacterial evolution. Reviewers This article was reviewed by Eugene V. Koonin, Nicolas Galtier, and Jeremy Selengut.

The eukaryotic 5' untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5' upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5'UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer.