An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.

New insights into other importantPublisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature06269Page 2 of 33 symbiotic functions including H 2 metabolism, CO 2 -reductive acetogenesis and N 2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.All known termite species form obligate, nutritional mutualisms with diverse gut microbial species found nowhere else in nature 3 . Despite nearly a century of study, however, science still has only a meagre understanding of the exact roles of the host and symbiotic microbiota in the complex processes of lignocellulose degradation and conversion. Especially conspicuous is our poor understanding of the hindgut communities of wood-feeding 'higher'termites, the most species-rich and abundant of all termite lineages 4 . Higher termites do not contain hindgut flagellate protozoa, which have long been known to be sources of cellulases and hemicellulases in the 'lower' termites. The host tissue of all wood-feeding termites is known to be the source of one cellulase, a single-domain glycohydrolase family 9 enzyme that is secreted and active in the anterior compartments of the gut tract 5 . Only in recent years has research provided support for a role of termite gut bacteria in the production of relevant hydrolytic enzymes. That evidence includes the observed tight attachment of bacteria to wood particles, the antibacterial sensitivity of particle-bound cellulase activity 2 , and the discovery of a gene encoding a novel endoxylanase (glycohydrolase family 11) from bacterial DNA harvested from the gut tract of a Nasutitermes species 6 . Here, in an effort to learn about gene-centred details relevant to the diverse roles of bacterial symbionts in these successful wood-degrading insects,we initiated a metagenomic analysis of a wood-feeding 'higher' termite hindgut community, performed a proteomic analysis with clarified gut fluid from the same sample, and examined a set of candidate enzymes identified during the course of the study for demonstrable cellulase activity.A nest of an arboreal species closely related to Nasutitermes ephratae and N. corniger ( Supplementary Fig. 1) was collected near Guápiles, Costa Rica. From worker specimens, luminal contents were sampled specifically from the largest hindgut compartment, the microbedense, microlitre-sized region alternatively known as the paunch or the third proctodeal segment (P3; Fig. 1a). In the interest of interpretive clarity, we specifically excluded sampling from and analysis of the microbiota attached to the P3 epithelium and the other distinct microbial communities associated with the other hindgut compartments.Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter DOI: 10.1038/nature06269Page 3 of 33Total community DNA from pooled P3 luminal contents was purified, cloned and sequenced. About 71 million base pairs of Sang...

The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in "defense islands" in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.

Arrays of clustered, regularly spaced short palindromic repeats (CRISPR) are widespread in the genomes of many bacteria and almost all archaea. These arrays are composed of direct repeats sized 24-47 bp separated by similarly sized non-repetitive sequences (spacers). It was recently experimentally shown that CRISPR arrays, along with a group of associated proteins, confer resistance to phage. Following exposure to phage, bacteria integrate new spacer sequences that are derived from the phage genome. Acquisition of these spacers enables the bacterial cell to shutdown the phage attack, presumably by an RNA-interference-like mechanism. This Progress discusses the structure and function of CRISPRs and the implications of this new antiviral mechanism in bacteria.2 Bacteriophages constitute the most populous life-forms on Earth 1 . In sea water, an environment in which phage abundance has been extensively studied, it has been estimated that there are 5-10 phage for every bacterial cell 2 . Despite being outnumbered by phage, bacteria proliferate and avoid extinction by using a battery of innate phageresistance mechanisms such as restriction enzymes and abortive infection 3 . In this Progress article we describe the CRISPR system, a recently discovered defence mechanism, which is remarkable because it confers acquired phage resistance in Bacteria and Archaea. A hallmark of this system are arrays of short direct repeats interspersed by non-repetitive spacer sequences, the so-called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Additional components of the system include CRISPRassociated (CAS) genes and a leader sequence (Fig. 1A). Brief history of CRISPR researchThe first report that described a CRISPR array, in 1987, was from Ishino et al. who found 14 repeats of 29bp interspersed by 32-33bp non-repeating spacer sequences 4,5 , adjacent to the isozyme converting alkaline phosphatase (iap) gene in Escherichia coli. In subsequent years similar CRISPR arrays were found in Mycobacterium tuberculosis 6 , Haloferax mediterranei 7 , Methanocaldococcus jannaschii 8 , Thermotoga maritima 9 and other bacteria and archaea. The accumulation of sequenced microbial genomes allowed genome-wide computational searches for CRISPRs (the first such analysis was carried out by Mojica et al. in 2000 10 ), and the most recent computational analyses revealed that CRISPRs are found in ~40% of bacterial and ~90% of archaeal genomes sequenced to date 11, 12 (Box 1).In parallel to the initial appreciation of the abundance of CRISPRs 13 , Jansen et al. identified four CRISPR-associated (CAS) genes that were almost always found adjacent to the repeat arrays 14 . Subsequent studies initiated by Koonin and colleagues 15, 16 and Haft et al. 17 uncovered 25-45 additional CAS genes appearing in close proximity to the arrays. The same set of genes is absent from genomes that lack CRISPRs.Several hypotheses for the function of CRISPRs have been proposed. Early in 1995 Mojica et al. suggested that the repeats were involved i...

Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses need to decide between the lytic and the lysogenic cycles, i.e., whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the spBeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a 6aa communication peptide that is released to the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We termed this communication system the “arbitrium” system, and further show that it is encoded by 3 phage genes: aimP, producing the peptide, aimR, the intracellular peptide receptor, and aimX, a negative regulator of lysogeny. The arbitrium system enables an offspring phage to communicate with its predecessors, i.e., to estimate the amount of recent prior infections and hence decide whether to employ the lytic or lysogenic cycle.

Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species' growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak to trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes.

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites while strictly avoiding self-antigens that would result in autoimmunity. The cellular machinery and complex signaling pathways that coordinate an effective immune response have generally been considered properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence-specific targeting of foreign nucleic acids was recently discovered in bacteria and archaea. Molecular vaccination in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into a repetitive locus in the host chromosome known as a CRISPR (clustered regularly interspaced short palindromic repeat). Here we review the mechanisms of CRISPR-mediated immunity and discuss the ecological and evolutionary implications of these adaptive defense systems.

An increasing number of eukaryotic genes are being found to have naturally occurring antisense transcripts. Here we study the extent of antisense transcription in the human genome by analyzing the public databases of expressed sequences using a set of computational tools designed to identify sense-antisense transcriptional units on opposite DNA strands of the same genomic locus. The resulting data set of 2,667 sense-antisense pairs was evaluated by microarrays containing strand-specific oligonucleotide probes derived from the region of overlap. Verification of specific cases by northern blot analysis with strand-specific riboprobes proved transcription from both DNA strands. We conclude that > or =60% of this data set, or approximately 1,600 predicted sense-antisense transcriptional units, are transcribed from both DNA strands. This indicates that the occurrence of antisense transcription, usually regarded as infrequent, is a very common phenomenon in the human genome. Therefore, antisense modulation of gene expression in human cells may be a common regulatory mechanism.