Molecular biology

The association of microRNAs with Argonaute proteins (AGOs) yields complexes regulating gene expression. Although bacterial and archaeal miRNAs show no sequence preference at their 5′ ends, eukaryotic miRNAs tend to have a 5′ U or A. Here the structure of the human AGO2 MID domain complexed with ribonucleotide monophosphates is solved, revealing specific interaction of UMP and AMP with a loop that discriminates against CMP or GMP, and explaining the observed preference.

The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme that can phosphorylate or dephosphorylate isocitrate dehydrogenase (ICDH) to either inactivate or activate it in response to environmental changes. Now the structures of AceK and the AceK–ICDH complex have been solved, revealing the conformational changes that occur when AceK changes from a kinase to a phosphatase and vice versa.

Proper functioning of the brain requires a balance between the formation of excitatory and inhibitory synapses, but how this is achieved during development is unclear. Here FGF22 and FGF7, two fibroblast growth factor cell–cell signalling molecules, are shown to promote the formation of excitatory and inhibitory synapses, respectively, through their effect on epilepsy in mice. These findings should inform other neurological and psychiatric disorders involving defects in synapse formation.

Proteins often comprise domains that can be distinguished as relatively separate regions in the three-dimensional structure. Communication between these domains is important for catalysis, regulation and folding, but how they communicate is largely unclear. Here, single-molecule optical tweezers were used to pull on a protein while monitoring the energetics of unfolding and refolding events in disparate regions. By comparing topological variations of the same protein, new rules of cooperation between domains were derived.

The initiation of protein synthesis requires the eukaryotic translation initiation factor (eIF) 2, which uses energy from the hydrolysis of GTP. Another factor, eIF5, accelerates the GTP-hydrolysing activity of eIF2. Here, two other roles for eIF5 have been defined. One involves stabilizing GDP, the product of GTP hydrolysis, on eIF2. In its other role, eIF5 works with phosphorylated eIF2 to inhibit the guanine-nucleotide exchange factor eIF2B. These results clarify our understanding of how the initiation of translation is regulated.

Here, a library of more than 300,000 chemicals was screened for activity against Plasmodium falciparum growing in red blood cells. Of these chemicals, 172 representative candidates were profiled in detail; one exemplar compound showed efficacy in a mouse model of malaria. The findings provide the scientific community with new starting points for drug discovery.

Toll-like receptors (TLRs) are crucial to innate immunity. Activation of these proteins, and of receptors for the pro-inflammatory cytokines IL-1 and IL-18, leads to the recruitment of adaptor proteins such as MyD88. These in turn interact with further proteins such as IRAK2 and IRAK4. The crystal structure of the MyD88–IRAK2–IRAK4 death domain complex is now reported, explaining how these three proteins cooperate in TLR/IL-1R signalling.

Staphylococcal superantigens can lead to toxic shock syndrome. They are encoded on pathogenicity islands and with the aid of helper phages can be excised and packaged into highly transmissable phage particles. Here it is shown that a specific, non-essential helper phage protein is responsible for derepression of the pathogenicity island, thereby providing the mechanism for the first step of its mobilization.

Traditional robots need to store internal representations of their goals and environment, and to coordinate sensing and the movement of components required in response. Individual molecules are limited in their ability to store complex information, but robotic behaviour can still be realized — as has now been shown with DNA walkers, which can carry out a sequence of actions such as 'start', 'follow', 'turn' and 'stop' that are programmed into the DNA landscape on which the walkers move.

Spider silk proteins are remarkably soluble when stored at high concentration and yet can be converted to extremely sturdy fibres, through unknown molecular mechanisms. Here, the X-ray structure of the amino-terminal domain of a silk protein is presented, revealing how evolutionarily conserved polar surfaces might control self-assembly as the pH is lowered along the spider's silk extrusion duct. Such a mechanism might be applicable to the design of versatile fibrous materials.

The main reason why tumours are not controlled by the immune system is that they do not express potent tumour rejection antigens. Tumour vaccination aims to provoke a response to any antigens that are expressed. Here, a new approach is described: nonsense-mediated messenger RNA decay in tumour cells is inhibited, leading to the expression of new antigens and to significant inhibition of tumour growth in mice.

Post-replicative repair (PRR) enables cells to bypass or overcome DNA damage during DNA replication. In eukaryotes, ubiquitylation of the replication clamp PCNA by components of the RAD6 pathway activates damage bypass. When this occurs has been debated. It is now shown that PRR can be postponed until much of the undamaged genome is replicated. Moreover, it seems that PRR occurs mainly by an error-prone process, with error-free bypass playing a minor role.

The coding capacity of the genome is greatly expanded by the process of alternative splicing, which enables a single gene to produce more than one distinct protein. Can the expression of these different proteins be predicted from sequence data? Here, modelling based on information theory has been used to develop a 'splicing code', which can predict, with good accuracy, tissue-dependent changes in alternative splicing.

The topologies of, and spatial relationships between, chromosomes are important but poorly understood. Here, a high-throughput method is used to study intra- and inter-chromosomal interactions in Saccharomyces cerevisiae. A map of the haploid genome is generated at kilobase resolution, and is used to construct a three-dimensional model of the yeast genome. The findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

Polycomb group (PcG) proteins are transcriptional repressors that modify chromatin and regulate important developmental genes. One PcG-associated, chromatin-modifying activity is an enzyme that ubiquitinates histone H2A of chromatin. Here, a fruitfly PcG complex that is associated with H2A deubiquitination, and thereby with gene repression, is identified. PcG-mediated gene silencing might thus involve a dynamic balance between ubiquitination and deubiquitination of H2A.

MicroRNAs, which regulate gene expression, are transcribed as longer sequences that are processed to produce the mature form. Two nuclease enzymes, Drosha and Dicer, are known to act sequentially to trim the microRNA to size. Here, however, a subset of microRNAs that includes miR-451, important for erythropoiesis, is found to be processed independently of Dicer. Rather, the Argonaute protein — part of the complex that aligns microRNA and messenger RNA — carries out the secondary cleavage.

Notch and Delta are transmembrane proteins that allow neighbouring cells to communicate during development. Here, quantitative time-lapse microscopy has been used to show that the response of Notch to Delta on a neighbouring cell is graded, whereas its response to Delta on the same cell is sharp and occurs at a fixed threshold. A mathematical model explores how this new design principle enhances the sharpness of developmental boundaries set by classical lateral inhibition.

Induced pluripotent stem cells (iPSCs) are generated by the enforced expression of particular transcription factors in somatic cells. The extent to which such cells are equivalent to embryonic stem (ES) cells is an open question. Here, genetically identical mouse ES cells and iPSCs have been compared; the overall expression patterns of messenger RNAs and microRNAs are the same, with the exception of a few transcripts encoded within an imprinted gene cluster on chromosome 12qF1.

SUMOylation is a post-translational protein modification that affects many eukaryotic cellular processes. It is shown here that cellular infection with Listeria monocytogenes induces degradation of one of the essential SUMOylation enzymes, Ubc9, through a mechanism that involves a bacterial toxin, listeriolysin O. This effect on SUMOylation may support efficient infection by Listeria.

DNA methylation is an important epigenetic mark in many eukaryotes. In Arabidopsis plants, small interfering RNAs bound to the Argonaute 4 (AGO4) protein can direct de novo DNA methylation and consequent gene silencing. Here, a new regulator of RNA-directed DNA methylation has been discovered. This protein, RDM1, is proposed to bind to methylated DNA and to function in the AGO4 effector complex.

Most human gene promoters are embedded within CpG islands that lack DNA methylation and coincide with sites at which histone H3 lysine 4 is trimethylated (H3K4me3 sites). Here, a zinc-finger protein, Cfp1, is found to be associated with non-methylated CpG islands and H3K4me3 sites throughout the genome in the mouse brain. A primary function of non-methylated CpG islands might be to genetically determine the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins.

Single-molecule studies allow biological processes to be examined one molecule at a time, as they occur. Here, zero-mode waveguides have been used to concentrate reactions in zeptolitre-sized volumes, making it possible to study real-time translocation by the ribosome. The binding of transfer RNAs (tRNAs) to the ribosome could be followed; the results show that tRNA release from the exit site is uncoupled from tRNA binding to the aminoacyl-tRNA site.

Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome. Here it is shown that lincRNAs in the HOX genetic loci are dysregulated during breast cancer progression in human cells, and that expression levels of the lincRNA called HOTAIR can predict whether a tumour will metastasize. Moreover, enforced expression of HOTAIR can lead to altered patterns of binding of the PRC2 protein to the genome.

Regulatory proteins bind non-coding DNA either at promoters (near to a gene's transcription start site) or at enhancers (far away). Binding at enhancers helps to bring the transcription enzyme RNA polymerase to promoters. Here, studies of some 12,000 enhancers that respond to electrical activity in neurons show that binding to enhancers also brings the polymerase to the enhancers themselves, where it transcribes a novel class of non-coding RNAs. Enhancers may thus be more similar to promoters than hitherto appreciated.

Most agents that generate breaks in DNA leave 'dirty ends' that cannot be joined immediately; instead, intervening steps are required to restore the integrity of nucleotides at the break. Here it is shown that the non-homologous end joining pathway requires a 5′-dRP/AP lyase activity to remove abasic sites at double-strand breaks. Surprisingly, this activity is catalysed by the Ku70 protein, which, together with its partner Ku86, had been thought only to recognize broken DNA ends and to recruit other factors that process ends.

Animals must detect water in their environment to stay alive, but the molecular basis for water detection has been unclear. Here the essential mediators of water-sensing and drinking in fruitflies have been identified: an ion channel of the degenerin/epithelial sodium channel family, and the sensory neurons that make it.

During embryonic development, blood vessels remodel in response to blood flow. Here, a genetic pathway is described through which this mechanosensory stimulus is integrated with early developmental signals to remodel vessels of the aortic arch in zebrafish. It is found that the flow-induced transcription factor klf2a is required to induce the expression of an endothelial-specific microRNA, activating signalling through the growth factor Vegf.

In meiotic cells paired homologues are joined by a set of crossovers known as a double Holliday junction (DHJ). Whether DHJs form during mitotic recombination has been unclear, as mitotic cells possess alternative repair pathways that would not require DHJ formation. Here it is demonstrated that mitotic and meiotic cells form similar DHJs, but that the levels in mitotic cells are approximately 10–fold lower, and show a preference for joints between sister chromatids rather than homologues. Consequently, in mitotic cells non–crossover outcomes are favoured.

To study the changes in chromatin structure that accompany zygotic genome activation and pluripotency during the maternal–zygotic transition (MZT), the genomic locations of histone H3 modifications and RNA polymerase II have been mapped during this transition in zebrafish embryos. H3 lysine 27 trimethylation and H3 lysine 4 trimethylation are only detected after MZT; evidence is provided that the bivalent chromatin domains found in cultured embryonic stem cells also exist in embryos.

The amino-terminal tails of histone proteins are subject to a variety of post-translational modifications; addition or removal of these 'marks' facilitates gene activation or silencing. Here, a mechanism is defined that modulates the activity of the dual-specificity histone demethylase LSD1 during androgen-dependent transcription. Androgen-dependent signalling through protein kinase C beta I leads to phosphorylation of a histone amino acid, which prevents demethylation of an adjacent amino acid by LSD1.

Female gametes in flowering plants develop from a meiotic division of a precursor cell followed by mitotic divisions of one of the resulting haploid cells to yield the gametophyte. Here, ARGONAUTE 9 (AGO9) — a protein involved in RNA interference — is identified as a factor required for specification of the gametophyte. AGO9 is found not in the cell destined to be the gametophyte, but in the neighbouring companion cells, suggesting that it functions in a non-cell-autonomous manner.

In response to oncogenic stress, the tumour suppressor ARF activates the p53 protein. ARF protein is highly stable in most human cell lines, so it has been thought that ARF activation occurs mainly at the level of transcription. Here, however, ARF is shown to be unstable in normal human cells but stable in cancer cells, through a transcription-independent mechanism. A ubiquitin ligase for ARF is identified and shown to promote ARF degradation in normal cells. This activity is prevented in cancer cells, stabilizing ARF.

An understanding of how fat cells (adipocytes) develop will contribute to our understanding of obesity. The differentiation of committed preadipocytes into adipocytes is known to be controlled by PPARγ and several other transcription factors. But what turns a cell into a preadipocyte? Here, the zinc-finger protein Zfp423 is identified as a transcriptional regulator of preadipocyte determination.

Evolution from one fitness peak to another must involve either transitions through intermediates of low fitness or skirting round the fitness valley through compensatory mutations elsewhere. Here, the base pairs in mitochondrial tRNA stems is used as a model to show that deep fitness valleys can be traversed. Transitions between AU and GC pairs have occurred during mammalian evolution without help from genetic drift or mutations elsewhere.

Endogenous retroviruses (ERVs) are widely dispersed in mammalian genomes, and are silenced in somatic cells by DNA methylation. Here, an ERV silencing pathway independent of DNA methylation is shown to operate in embryonic stem cells. The pathway involves the histone H3K9 methyltransferase ESET and might be important for ERV silencing during the stages in embryogenesis when DNA methylation is reprogrammed.

Although new amino acids with desirable properties can be devised, only a few have been successfully introduced into proteins by the cellular machinery. Even then, only one type of unnatural amino acid can be added to a given protein. Here, a new system has been designed that could allow the incorporation of up to 200 novel amino acids. The system involves an orthogonal ribosome that uses quadruplet — rather than triplet — codons, as well as orthogonal tRNA synthetase–tRNA pairs.

The 'thermodynamic hypothesis' proposes that the sequence of a biological macromolecule defines its folded, active structure as a global energy minimum in the folding landscape; however, it is not clear whether there is only one global minimum or several local minima corresponding to active conformations. Here, using single-molecule experiments, an RNA enzyme is shown to fold into multiple distinct native states that interconvert.

Recent work suggests that microRNAs might have been important in the evolution of complexity in multicellular animals. Here it is shown that the most ancient known microRNA, miR–100, was initially active in neurosecretory cells around the mouth. Other highly conserved varieties were first present in specific tissues and organ systems. Thus, microRNA expression was initially restricted to an ancient set of ancient animal cell types and tissues.

The extent of epigenetic reprogramming in mammalian primordial germ cells (PGCs) and in early embryos, and its molecular mechanisms, are poorly understood. DNA methylation profiling in PGCs now reveals a genome–wide erasure of methylation, with female PGCs being less methylated than male ones. A deficiency of the cytidine deaminase AID interferes with the genome–wide erasure of DNA methylation, indicating that AID has a critical function in epigenetic reprogramming.

Although cyclin D1 is frequently overexpressed in human cancers, the full range of its functions in normal development and oncogenesis is unclear. Here, tagged cyclin D1 knock-in mouse strains are developed to allow a search for cyclin D1-binding proteins in different mouse organs using high-throughput mass spectrometry. The results show that, in addition to its established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development.

Progenitor cells sustain the capacity of self-renewing tissues for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation is one potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through cell divisions. The DNA methyltransferase 1 and other regulators of DNA methylation are now shown to be essential for epidermal progenitor cell function.

Much of the mammalian genome is derived from retroelements, a significant proportion of which are endogenous retroviruses (ERVs). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation, but the initiators of this process are largely unknown. Here, deletion of KAP1 is shown to lead to a marked upregulation of a range of ERVs in mouse embryonic stem cells and in early embryos.

Polycomb proteins have a key role in regulating the expression of genes essential for development, differentiation and maintenance of cell fates. Here, Polycomb repressive complex 2 (PRC2) is shown to form a complex with JARID2, a Jumonji domain protein. JARID2 is required for the binding of Polycomb proteins to target genes in embryonic stem cells as well as for the proper differentiation of ES cells.

Rho is a general transcription termination factor in bacteria, but the mechanism by which it disrupts the RNA polymerase (RNAP) elongation complex is unknown. Here, Rho is shown to bind tightly to the RNAP throughout the transcription cycle, with the formation of the RNAP–Rho complex being crucial for termination. Furthermore, RNAP is proposed to have an active role in Rho termination through an allosteric mechanism.

Little is known about the recent evolution of the Y chromosome because only the human Y chromosome has been fully sequenced. The sequencing of the male-specific region of the Y chromosome (MSY) in the chimpanzee and comparison between the MSYs of the two species now reveals that they differ radically in sequence structure and gene content, indicating rapid evolution over the past 6 million years.