Mali Salmon-Divon

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq

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 N6-methyladenosine (m6A) 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 m6A modification landscape in a transcriptome-wide manner, using a novel approach, m6A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m6A 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 m6A 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 m6A has a fundamental role in regulation of gene expression.

m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation

mRNA modification regulates pluripotency When stem cells progress from an embryonic pluripotent state toward a particular lineage, molecular switches dismantle the transcription factor network that keeps the cell pluripotent. Geula et al. now show that N6-methyladenosine (m6A), a messenger RNA (mRNA) modification present on transcripts of pluripotency factors, drives this transition. Methylation destabilized mRNA transcripts and limited their translation efficiency, which promoted the timely decay of naïve pluripotency. This m6A methylation was also critical for mammalian development. Science, this issue p. 1002 A messenger RNA epigenetic modification regulates stem cell progression from the pluripotent to the differentiated state. Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

The shaping and functional consequences of the microRNA landscape in breast cancer.

MicroRNAs (miRNAs) show differential expression across breast cancer subtypes, and have both oncogenic and tumour-suppressive roles. Here we report the miRNA expression profiles of 1,302 breast tumours with matching detailed clinical annotation, long-term follow-up and genomic and messenger RNA expression data. This provides a comprehensive overview of the quantity, distribution and variation of the miRNA population and provides information on the extent to which genomic, transcriptional and post-transcriptional events contribute to miRNA expression architecture, suggesting an important role for post-transcriptional regulation. The key clinical parameters and cellular pathways related to the miRNA landscape are characterized, revealing context-dependent interactions, for example with regards to cell adhesion and Wnt signalling. Notably, only prognostic miRNA signatures derived from breast tumours devoid of somatic copy-number aberrations (CNA-devoid) are consistently prognostic across several other subtypes and can be validated in external cohorts. We then use a data-driven approach to seek the effects of miRNAs associated with differential co-expression of mRNAs, and find that miRNAs act as modulators of mRNA–mRNA interactions rather than as on–off molecular switches. We demonstrate such an important modulatory role for miRNAs in the biology of CNA-devoid breast cancers, a common subtype in which the immune response is prominent. These findings represent a new framework for studying the biology of miRNAs in human breast cancer.

The dynamic N 1 -methyladenosine methylome in eukaryotic messenger RNA

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m1A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA.

NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression

Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage‐specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD‐mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene‐specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.

Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

N6-methyladenosine–sequencing (m6A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m6A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation–sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m6A between and within gene transcripts. When applied to human and mouse transcriptomes, m6A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m6A. The protocol can be completed within ∼9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).

Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules

The ability of murine NK cells to reject cells lacking self MHC class I expression results from an in vivo education process. To study the impact of individual MHC class I alleles on this process, we generated mice expressing single MHC class I alleles (Kb, Db, Dd, or Ld) or combinations of two or more alleles. All single MHC class I mice rejected MHC class I–deficient cells in an NK cell–dependent way. Expression of Kb or Dd conveyed strong rejection of MHC class I–deficient cells, whereas the expression of Db or Ld resulted in weaker responses. The educating impact of weak ligands (Db and Ld) was further attenuated by the introduction of additional MHC class I alleles, whereas strong ligands (Kb and Dd) maintained their educating impact under such conditions. An analysis of activating and inhibitory receptors in single MHC class I mice suggested that the educating impact of a given MHC class I molecule was controlled both by the number of NK cells affected and by the strength of each MHC class I–Ly49 receptor interaction, indicating that NK cell education may be regulated by a combination of qualitative and quantitative events.

PeakAnalyzer: Genome-wide annotation of chromatin binding and modification loci

BackgroundFunctional genomic studies involving high-throughput sequencing and tiling array applications, such as ChIP-seq and ChIP-chip, generate large numbers of experimentally-derived signal peaks across the genome under study. In analyzing these loci to determine their potential regulatory functions, areas of signal enrichment must be considered relative to proximal genes and regulatory elements annotated throughout the target genome Regions of chromatin association by transcriptional regulators should be distinguished as individual binding sites in order to enhance downstream analyses, such as the identification of known and novel consensus motifs.ResultsPeakAnalyzer is a set of high-performance utilities for the automated processing of experimentally-derived peak regions and annotation of genomic loci. The programs can accurately subdivide multimodal regions of signal enrichment into distinct subpeaks corresponding to binding sites or chromatin modifications, retrieve genomic sequences encompassing the computed subpeak summits, and identify positional features of interest such as intersection with exon/intron gene components, proximity to up- or downstream transcriptional start sites and cis-regulatory elements. The software can be configured to run either as a pipeline component for high-throughput analyses, or as a cross-platform desktop application with an intuitive user interface.ConclusionsPeakAnalyzer comprises a number of utilities essential for ChIP-seq and ChIP-chip data analysis. High-performance implementations are provided for Unix pipeline integration along with a GUI version for interactive use. Source code in C++ and Java is provided, as are native binaries for Linux, Mac OS X and Windows systems.

ALA induced photodynamic effects on Gram positive and negative bacteria

In the present study we examined the production of high amounts of porphyrins upon induction by delta-aminolevulinic acid (ALA) in 9 bacterial strains. This was performed by solely inducing the porphyrin biosynthesis pathway. Four of the strains were Gram positive bacteria and five were Gram negative strains. All strains, except Streptococcus faecalis, produced porphyrins when incubated in PBS with 0.38 mM ALA for 4 h. Excess porphyrin production was excreted to the medium. Gram positive bacteria exhibited fluorescent emission peaks at 622 nm for the endogenous and 617 nm for the excreted porphyrins. Gram negative bacteria exhibited a 630 nm emission peak for the endogenous and a 615 nm emission peak for the excreted extracellular porphyrins. Upon illumination of the ALA induced Staphylococcal strains with 407-420 nm blue light, a decrease of five orders of magnitude was demonstrated with a light dose of 50 J cm(-2). Total eradication of the Staphylococcal strains could be achieved with a 100 J cm(-2) dose, which resulted in a decrease in viability of seven orders of magnitude. The viability of all the induced Gram negative strains and B. cereus decreased by one or two orders of magnitude upon illumination with 50 and 100 J cm(-2), respectively. This difference in the photoinactivation rate was found to be due to the distribution and amounts of the various porphyrins in the bacterial strains. The predominant porphyrin in the Staphylococcal strains was coproporphyrin (68.3-74.6%). In the Gram negative strains there was no predominant porphyrin and the porphyrins found were mostly 5-carboxyporphyrin, uroporphyrin, 7- carboxyporphyrin, coproporphyrin and protoporphyrin. In the B. cereus(Gram positive) strain the predominant porphyrin was uroporphyrin (75.8%). Although the total production of porphyrins in the Gram negative bacteria was higher than in the Staphylococcal strains, the amount of coproporphyrin produced by the latter was twice to three times higher than in the Gram negative strains. The extracellular excreted porphyrins did not contribute to the photoinactivation in any of the tested strains. Significant decreases in the Na(+) and K(+) content were detected in induced S. aureus after illumination while only small changes were observed in E. coli B. The green fluorescent protein within the cytoplasm of induced E. coli strains was only partially disrupted (by 60% only). These results indicate a partial yield of the effects generated by (1)O(2) radicals resulting from the photoinactivation of Gram negative bacteria and a successful generation of the same effects in the Staphylococcal strains.

Mechanistic aspects of Escherichia coli photodynamic inactivation by cationic tetra-meso(N-methylpyridyl)porphine

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been studied in bacteria expressing the reporter protein GFP, following transfection with wild type pGFP plasmid and treatment with the hydrophilic cationic sensitizer tetra-meso(N-methyl-4-pyridyl)porphine tetratosylate (TMPyP). Cell survival and morphology during PDI were correlated with plasmid-GFP degradation in comparison to DNA and RNA strand-breaks, while photobleaching of the GFP chromophore was used to monitor protein photodamage. Singlet oxygen generated upon TMPyP photoactivation interacted with target nucleic acid polymers in a drug-and light-dose dependent manner. The hierarchy and cascade of the photodamage was in the order: genomic-DNA > total RNA > plasmid-DNA, as revealed by specific extraction and agarose electrophoresis. The notable resistance of the plasmid DNA in comparison to genomic DNA has implications for PDI of antibiotic-resistant bacteria. Re-growth of the treated cells in fresh medium showed structural features of an SOS response. Under these conditions, DNA repair machinery was initiated by typical alignment of DNA-protein co-aggregates accompanied by lateral assembly of ribosomes, apart from damaged DNA-arrays, as depicted by electron microscopy. GFP-TMPyP interactions were demonstrated by double green and red fluorescence on electrophoresis plates analyzed by spectral imaging. Photobleaching measurements revealed specific GFP photodamage directly related to PDI of the E. coli. The kinetics of both the GFP photobleaching and the K(+) efflux, representing photodamage to cytosolic proteins and membrane damage, respectively, were found to be similar. The survival curves were correlated to chromosomal degradation and ultrastructural damage. We conclude that TMPyP-dependent PDI of E. coli is primarily dependent on genomic DNA photodamage rather than on protein or membrane malfunctions.