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L-lactate modifies proteins through lactylation 1 , but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular L-lactate sensors required for L-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to L-lactate with micromolar affinity and they directly catalyse L-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to L-lactate, AARS2 associates with cyclic GMP-AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of L-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular L-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Sequence-directed variations in the canonical DNA double helix structure that retain Watson-Crick base-pairing have important roles in DNA recognition, topology and nucleosome positioning. By using nuclear magnetic resonance relaxation dispersion spectroscopy in concert with steered molecular dynamics simulations, we have observed transient sequence-specific excursions away from Watson-Crick base-pairing at CA and TA steps inside canonical duplex DNA towards low-populated and short-lived A•T and G•C Hoogsteen base pairs. The observation of Hoogsteen base pairs in DNA duplexes specifically bound to transcription factors and in damaged DNA sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base pairs as a means of expanding its structural complexity beyond that which can be achieved based on Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient low-populated nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5′-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications. [ABSTRACT FROM AUTHOR]

DNA is famous as the instruction manual of life--the multi-billion-base-pair data tape that directs how a fertilized egg turns into the specific cells, tissues and organs of, say, a sharp-eyed [...]

In photosynthetic organisms, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins. [ABSTRACT FROM AUTHOR]

The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis. [ABSTRACT FROM AUTHOR]

Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNAAla selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3·U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3·U70 and its A3·U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3·U70, widening the major groove. The geometry difference between G3·U70 and A3·U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNAla with G3·U70 is oriented to the reactive route that reaches the active site, whereas that of the A3·U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100 -fold difference in kcat. [ABSTRACT FROM AUTHOR]

Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet. [ABSTRACT FROM AUTHOR]

Only a very small fraction of long noncoding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but the absence of lncRNA annotations in non-model organisms has precluded comparative analyses. Here we present a large-scale evolutionary study of lncRNA repertoires and expression patterns, in 11 tetrapod species. We identify approximately 11,000 primate-specific lncRNAs and 2,500 highly conserved lncRNAs, including approximately 400 genes that are likely to have originated more than 300 million years ago. We find that lncRNAs, in particular ancient ones, are in general actively regulated and may function predominantly in embryonic development. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network suggests potential functions for lncRNAs in fundamental processes such as spermatogenesis and synaptic transmission, but also in more specific mechanisms such as placenta development through microRNA production. [ABSTRACT FROM AUTHOR]

In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3′ untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation. [ABSTRACT FROM AUTHOR]

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits. [ABSTRACT FROM AUTHOR]

Recent reports have described an intricate interplay among diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs. These RNA transcripts act as competing endogenous RNAs (ceRNAs) or natural microRNA sponges - they communicate with and co-regulate each other by competing for binding to shared microRNAs, a family of small non-coding RNAs that are important post-transcriptional regulators of gene expression. Understanding this novel RNA crosstalk will lead to significant insight into gene regulatory networks and have implications in human development and disease. [ABSTRACT FROM AUTHOR]

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains 1-4 . Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present 5-12 . Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref.  13 ) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation. [ABSTRACT FROM AUTHOR]

During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine-purine base pairs. Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNASer bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine-purine base pairs at the second and third positions of the codon, which show an unusual Watson-Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs. [ABSTRACT FROM AUTHOR]

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet. [ABSTRACT FROM AUTHOR]

The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription. [ABSTRACT FROM AUTHOR]

Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect messenger RNA stability, localization and translation, or produce truncated proteins that differ in localization or function. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed. Here, by jointly determining both transcript ends for millions of RNA molecules, we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries seems to be the rule rather than the exception, even within a single population of yeast cells. Over 26 major transcript isoforms per protein-coding gene were expressed in yeast. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, approximately 70% of genes express alternative isoforms that vary in post-transcriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution and phenotypic diversity between single cells. These data also indicate that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes. [ABSTRACT FROM AUTHOR]

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis. [ABSTRACT FROM AUTHOR]

Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes. Codon usage is also implicated in the control of transcription, splicing and RNA structure. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function. [ABSTRACT FROM AUTHOR]

In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination. [ABSTRACT FROM AUTHOR]

The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. [ABSTRACT FROM AUTHOR]

Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the 'oncometabolite' R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML. [ABSTRACT FROM AUTHOR]

Novel protein-coding genes can arise either through re-organization of pre-existing genes or de novo. Processes involving re-organization of pre-existing genes, notably after gene duplication, have been extensively described. In contrast, de novo gene birth remains poorly understood, mainly because translation of sequences devoid of genes, or 'non-genic' sequences, is expected to produce insignificant polypeptides rather than proteins with specific biological functions. Here we formalize an evolutionary model according to which functional genes evolve de novo through transitory proto-genes generated by widespread translational activity in non-genic sequences. Testing this model at the genome scale in Saccharomyces cerevisiae, we detect translation of hundreds of short species-specific open reading frames (ORFs) located in non-genic sequences. These translation events seem to provide adaptive potential, as suggested by their differential regulation upon stress and by signatures of retention by natural selection. In line with our model, we establish that S. cerevisiae ORFs can be placed within an evolutionary continuum ranging from non-genic sequences to genes. We identify ?1,900 candidate proto-genes among S. cerevisiae ORFs and find that de novo gene birth from such a reservoir may be more prevalent than sporadic gene duplication. Our work illustrates that evolution exploits seemingly dispensable sequences to generate adaptive functional innovation. [ABSTRACT FROM AUTHOR]

All cancers carry somatic mutations in their genomes. A subset, known as driver mutations, confer clonal selective advantage on cancer cells and are causally implicated in oncogenesis, and the remainder are passenger mutations. The driver mutations and mutational processes operative in breast cancer have not yet been comprehensively explored. Here we examine the genomes of 100 tumours for somatic copy number changes and mutations in the coding exons of protein-coding genes. The number of somatic mutations varied markedly between individual tumours. We found strong correlations between mutation number, age at which cancer was diagnosed and cancer histological grade, and observed multiple mutational signatures, including one present in about ten per cent of tumours characterized by numerous mutations of cytosine at TpC dinucleotides. Driver mutations were identified in several new cancer genes including AKT2, ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1 and TBX3. Among the 100 tumours, we found driver mutations in at least 40 cancer genes and 73 different combinations of mutated cancer genes. The results highlight the substantial genetic diversity underlying this common disease. [ABSTRACT FROM AUTHOR]

Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids. Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1), which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting. [ABSTRACT FROM AUTHOR]

During protein synthesis, the ribosome accurately selects transfer RNAs (tRNAs) in accordance with the messenger RNA (mRNA) triplet in the decoding centre. tRNA selection is initiated by elongation factor Tu, which delivers tRNA to the aminoacyl tRNA-binding site (A site) and hydrolyses GTP upon establishing codon-anticodon interactions in the decoding centre. At the following proofreading step the ribosome re-examines the tRNA and rejects it if it does not match the A codon. It was suggested that universally conserved G530, A1492 and A1493 of 16S ribosomal RNA, critical for tRNA binding in the A site, actively monitor cognate tRNA, and that recognition of the correct codon-anticodon duplex induces an overall ribosome conformational change (domain closure). Here we propose an integrated mechanism for decoding based on six X-ray structures of the 70S ribosome determined at 3.1-3.4?Å resolution, modelling cognate or near-cognate states of the decoding centre at the proofreading step. We show that the 30S subunit undergoes an identical domain closure upon binding of either cognate or near-cognate tRNA. This conformational change of the 30S subunit forms a decoding centre that constrains the mRNA in such a way that the first two nucleotides of the A codon are limited to form Watson-Crick base pairs. When U·G and G·U mismatches, generally considered to form wobble base pairs, are at the first or second codon-anticodon position, the decoding centre forces this pair to adopt the geometry close to that of a canonical C·G pair. This by itself, or with distortions in the codon-anticodon mini-helix and the anticodon loop, causes the near-cognate tRNA to dissociate from the ribosome. [ABSTRACT FROM AUTHOR]

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (?140?mm?Hg systolic blood pressure or??90?mm?Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention. [ABSTRACT FROM AUTHOR]

Structural variation is widespread in mammalian genomes and is an important cause of disease, but just how abundant and important structural variants (SVs) are in shaping phenotypic variation remains unclear. Without knowing how many SVs there are, and how they arise, it is difficult to discover what they do. Combining experimental with automated analyses, we identified 711,920 SVs at 281,243 sites in the genomes of thirteen classical and four wild-derived inbred mouse strains. The majority of SVs are less than 1?kilobase in size and 98% are deletions or insertions. The breakpoints of 160,000 SVs were mapped to base pair resolution, allowing us to infer that insertion of retrotransposons causes more than half of SVs. Yet, despite their prevalence, SVs are less likely than other sequence variants to cause gene expression or quantitative phenotypic variation. We identified 24 SVs that disrupt coding exons, acting as rare variants of large effect on gene function. One-third of the genes so affected have immunological functions. [ABSTRACT FROM AUTHOR]

All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (?), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to ? within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ?AA and ?AG code for serine and threonine, whereas ?GA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code. [ABSTRACT FROM AUTHOR]

The article offers information on the success of biologists and researchers to in copying an existing genetic code, but were not yet commercialized. It notes the significance of technology to help researchers work on several genes at a time. It mentions the technology created by the Venter Institute to build and transplant a genome which took them for 15 years. It suggests researchers to be able to copy natural genomes before creating new ones.

The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer. Here we show that casein kinase Iα (CKIα), a component of the β-catenin-destruction complex, is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKIα) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKIα-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation. The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression; however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis in CKIα-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53 or its target gene p21 (also known as Waf1, Cip1, Sdi1 and Cdkn1a) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKIα caused a highly invasive carcinoma, indicating that CKIα functions as a tumour suppressor when p53 is inactivated. We identified a set of genes (the p53-suppressed invasiveness signature, PSIS) that is activated by the loss of both p53 and CKIα and which probably accounts for the brisk induction of invasiveness. PSIS transcription and tumour invasion were suppressed by p21, independently of cell cycle control. Restraining tissue invasion through suppressing PSIS expression is thus a novel tumour-suppressor function of wild-type p53. [ABSTRACT FROM AUTHOR]

The sequence of the human genome has dramatically accelerated biomedical research. Here I explore its impact, in the decade since its publication, on our understanding of the biological functions encoded in the genome, on the biological basis of inherited diseases and cancer, and on the evolution and history of the human species. I also discuss the road ahead in fulfilling the promise of genomics for medicine. [ABSTRACT FROM AUTHOR]

The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs. [ABSTRACT FROM AUTHOR]

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase–tRNACUA (MjTyrRS–tRNACUA) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase–tRNACUA (MbPylRS–tRNACUA) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase–tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase–tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells. [ABSTRACT FROM AUTHOR]

Ca2+ channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of CaV1–2 Ca2+ channels are regulated by a single CaM constitutively complexed with channels. The Ca2+-free form of CaM (apoCaM) is already pre-associated with the isoleucine–glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca2+ binding to this ‘resident’ CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel–CaM coordination could arise from the absence of Ca2+ regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca2+ feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca2+ channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of CaV1.3 and CaV1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca2+ feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca2+ entry through channels is broadly significant for Ca2+ homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca2+ channels. [ABSTRACT FROM AUTHOR]

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code. [ABSTRACT FROM AUTHOR]

Recent advances in next generation sequencing have made it possible to precisely characterize all somatic coding mutations that occur during the development and progression of individual cancers. Here we used these approaches to sequence the genomes (>43-fold coverage) and transcriptomes of an oestrogen-receptor-α-positive metastatic lobular breast cancer at depth. We found 32 somatic non-synonymous coding mutations present in the metastasis, and measured the frequency of these somatic mutations in DNA from the primary tumour of the same patient, which arose 9 years earlier. Five of the 32 mutations (in ABCB11, HAUS3, SLC24A4, SNX4 and PALB2) were prevalent in the DNA of the primary tumour removed at diagnosis 9 years earlier, six (in KIF1C, USP28, MYH8, MORC1, KIAA1468 and RNASEH2A) were present at lower frequencies (1–13%), 19 were not detected in the primary tumour, and two were undetermined. The combined analysis of genome and transcriptome data revealed two new RNA-editing events that recode the amino acid sequence of SRP9 and COG3. Taken together, our data show that single nucleotide mutational heterogeneity can be a property of low or intermediate grade primary breast cancers and that significant evolution can occur with disease progression. [ABSTRACT FROM AUTHOR]

Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions (‘exomes’), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman–Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact. [ABSTRACT FROM AUTHOR]

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes. [ABSTRACT FROM AUTHOR]

Susceptibility to Crohn’s disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn’s disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn’s disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn’s disease patients carrying the Crohn’s disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn’s disease patients homozygous for the ATG16L1 Crohn’s disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn’s disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells. [ABSTRACT FROM AUTHOR]

The article discusses the significant role played by RNA in the field of molecular biology. It reveals that several studies were conducted by molecular biologists to assess the function of RNA in genetic transcription and cellular activities. It also discusses the important function played by long non-coding RNA in genetics, which include gene silencing, its ability to control protein-coding gene, and enables cells to know their functions in the human body. However, the author asserts that RNA sequencing is one of the challenges encountered by scientists in evaluating its importance which does not provide adequate information concerning its function.

Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated. [ABSTRACT FROM AUTHOR]

Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or ‘evolutionary signatures’, dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies. [ABSTRACT FROM AUTHOR]

Histone methylation is crucial for regulating chromatin structure, gene transcription and the epigenetic state of the cell. LSD1 is a lysine-specific histone demethylase that represses transcription by demethylating histone H3 on lysine 4 (ref. 1). The LSD1 complex contains a number of proteins, all of which have been assigned roles in events upstream of LSD1-mediated demethylation apart from BHC80 (also known as PHF21A), a plant homeodomain (PHD) finger-containing protein. Here we report that, in contrast to the PHD fingers of the bromodomain PHD finger transcription factor (BPTF) and inhibitor of growth family 2 (ING2), which bind methylated H3K4 (H3K4me3), the PHD finger of BHC80 binds unmethylated H3K4 (H3K4me0), and this interaction is specifically abrogated by methylation of H3K4. The crystal structure of the PHD finger of BHC80 bound to an unmodified H3 peptide has revealed the structural basis of the recognition of H3K4me0. Knockdown of BHC80 by RNA inhibition results in the de-repression of LSD1 target genes, and this repression is restored by the reintroduction of wild-type BHC80 but not by a PHD-finger mutant that cannot bind H3. Chromatin immunoprecipitation showed that BHC80 and LSD1 depend reciprocally on one another to associate with chromatin. These findings couple the function of BHC80 to that of LSD1, and indicate that unmodified H3K4 is part of the ‘histone code’. They further raise the possibility that the generation and recognition of the unmodified state on histone tails in general might be just as crucial as post-translational modifications of histone for chromatin and transcriptional regulation. [ABSTRACT FROM AUTHOR]