Global mRNA abundance depends on the balance of synthesis and decay of a population of mRNAs. To account for this balance during activation of T cells, we used metabolic labeling to quantify the contributions of RNA transcription and decay over a 4 h time course during activation of leukemia-derived Jurkat T cells. While prior studies suggested more than half of the changes in mRNA abundance were due to RNA stability, we found a smaller but more interesting population of mRNAs changed stability. These mRNAs clustered into functionally related subpopulations that included replicative histones, ribosomal biogenesis and cell motility functions. We then applied a novel analysis based on integrating global protein-RNA binding with concurrent changes in RNA stability at specific time points following activation. This analysis demonstrated robust stabilization of mRNAs by the HuR RNA-binding protein 4 h after activation. Our unexpected findings demonstrate that the temporal regulation of mRNA stability coordinates vital cellular pathways and is in part controlled by the HuR RNA binding protein in Jurkat T cells following activation.

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.

Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes.

To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA(Lys) UUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.

Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis.

The Med2, Med3 and Med15 proteins form a heterotrimeric subdomain in the budding yeast Mediator complex. This Med15 module is an important target for many gene specific transcription activators. A previous proteome wide screen in yeast identified Med3 as a protein with priogenic potential. In the present work, we have extended this observation and demonstrate that both Med3 and Med15 form amyloid-like protein aggregates under H2O2 stress conditions. Amyloid formation can also be stimulated by overexpression of Med3 or of a glutamine-rich domain present in Med15, which in turn leads to loss of the entire Med15 module from Mediator and a change in stress response. In combination with genome wide transcription analysis, our data demonstrate that amyloid formation can change the subunit composition of Mediator and thereby influence transcriptional output in budding yeast.

A key aspect of RNA secondary structure prediction is the identification of novel functional elements. This is a challenging task because these elements typically are embedded in longer transcripts where the borders between the element and flanking regions have to be defined. The flanking sequences impact the folding of the functional elements both at the level of computational analyses and when the element is extracted as a transcript for experimental analysis. Here, we analyze how different flanking region lengths impact folding into a constrained structure by computing probabilities of folding for different sizes of flanking regions. Our method, RNAcop (RNA context optimization by probability), is tested on known and de novo predicted structures. In vitro experiments support the computational analysis and suggest that for a number of structures, choosing proper lengths of flanking regions is critical. RNAcop is available as web server and stand-alone software via http://rth.dk/resources/rnacop.

There are currently 151 plants with draft genomes available but levels of functional annotation for putative protein products are low. Therefore, accurate computational predictions are essential to annotate genomes in the first instance, and to provide focus for the more costly and time consuming functional assays that follow. DNA-binding proteins are an important class of proteins that require annotation, but current computational methods are not applicable for genome wide predictions in plant species. Here, we explore the use of species and lineage specific models for the prediction of DNA-binding proteins in plants. We show that a species specific support vector machine model based on Arabidopsis sequence data is more accurate (accuracy 81%) than a generic model (74%), and based on this we develop a plant specific model for predicting DNA-binding proteins. We apply this model to the tomato proteome and demonstrate its ability to perform accurate high-throughput prediction of DNA-binding proteins. In doing so, we have annotated 36 currently uncharacterised proteins by assigning a putative DNA-binding function. Our model is publically available and we propose it be used in combination with existing tools to help increase annotation levels of DNA-binding proteins encoded in plant genomes.

In Paramecium, the regeneration of a functional somatic genome at each sexual event relies on the elimination of thousands of germline DNA sequences, known as Internal Eliminated Sequences (IESs), from the zygotic nuclear DNA. Here, we provide evidence that IESs' length and sub-terminal bases jointly modulate IES excision by affecting DNA conformation in P. tetraurelia. Our study reveals an excess of complementary base pairing between IESs' sub-terminal and contiguous sites, suggesting that IESs may form DNA loops prior to cleavage. The degree of complementary base pairing between IESs' sub-terminal sites (termed Cin-score) is positively associated with IES length and is shaped by natural selection. Moreover, it escalates abruptly when IES length exceeds 45 nucleotides (nt), indicating that only sufficiently large IESs may form loops. Finally, we find that IESs smaller than 46 nt are favored targets of the cellular surveillance systems, presumably because of their relatively inefficient excision. Our findings extend the repertoire of cis-acting determinants for IES recognition/excision and provide unprecedented insights into the distinct selective pressures that operate on IESs and somatic DNA regions. This information potentially moves current models of IES evolution and of mechanisms of IES recognition/excision forward.

Smad proteins are important intracellular mediators of TGF-β signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 domain contacts the GC-rich site with the same β-hairpin, but the detailed interaction modes are different. Conserved β-hairpin residues make base specific contacts with the minimal GC-rich site, 5'-GGC-3'. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 domain with the DNA targets.

On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.

The 22-mer c-kit promoter sequence folds into a parallel-stranded quadruplex with a unique structure, which has been elucidated by crystallographic and NMR methods and shows a high degree of structural conservation. We have carried out a series of extended (up to 10 μs long, ∼50 μs in total) molecular dynamics simulations to explore conformational stability and loop dynamics of this quadruplex. Unfolding no-salt simulations are consistent with a multi-pathway model of quadruplex folding and identify the single-nucleotide propeller loops as the most fragile part of the quadruplex. Thus, formation of propeller loops represents a peculiar atomistic aspect of quadruplex folding. Unbiased simulations reveal μs-scale transitions in the loops, which emphasizes the need for extended simulations in studies of quadruplex loops. We identify ion binding in the loops which may contribute to quadruplex stability. The long lateral-propeller loop is internally very stable but extensively fluctuates as a rigid entity. It creates a size-adaptable cleft between the loop and the stem, which can facilitate ligand binding. The stability gain by forming the internal network of GA base pairs and stacks of this loop may be dictating which of the many possible quadruplex topologies is observed in the ground state by this promoter quadruplex.

Establishment and differentiation of mammary alveoli during pregnancy are controlled by prolactin through the transcription factors STAT5A and STAT5B (STAT5), which also regulate temporal activation of mammary signature genes. This study addressed the question whether the methyltransferase and transcriptional co-activator EZH2 controls the differentiation clock of mammary epithelium. Ablation of Ezh2 from mammary stem cells resulted in precocious differentiation of alveolar epithelium during pregnancy and the activation of mammary-specific STAT5 target genes. This coincided with enhanced occupancy of these loci by STAT5, EZH1 and RNA Pol II. Limited activation of differentiation-specific genes was observed in mammary epithelium lacking both EZH2 and STAT5, suggesting a modulating but not mandatory role for STAT5. Loss of EZH2 did not result in overt changes in genome-wide and gene-specific H3K27me3 profiles, suggesting compensation through enhanced EZH1 recruitment. Differentiated mammary epithelia did not form in the combined absence of EZH1 and EZH2. Transplantation experiments failed to demonstrate a role for EZH2 in the activity of mammary stem and progenitor cells. In summary, while EZH1 and EZH2 serve redundant functions in the establishment of H3K27me3 marks and the formation of mammary alveoli, the presence of EZH2 is required to control progressive differentiation of milk secreting epithelium during pregnancy.

Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyper-phosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1-phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells.

De-novo motif search is a frequently applied bioinformatics procedure to identify and prioritize recurrent elements in sequences sets for biological investigation, such as the ones derived from high-throughput differential expression experiments. Several algorithms have been developed to perform motif search, employing widely different approaches and often giving divergent results. In order to maximize the power of these investigations and ultimately be able to draft solid biological hypotheses, there is the need for applying multiple tools on the same sequences and merge the obtained results. However, motif reporting formats and statistical evaluation methods currently make such an integration task difficult to perform and mostly restricted to specific scenarios. We thus introduce here the Dynamic Motif Integration Toolkit (DynaMIT), an extremely flexible platform allowing to identify motifs employing multiple algorithms, integrate them by means of a user-selected strategy and visualize results in several ways; furthermore, the platform is user-extendible in all its aspects. DynaMIT is freely available at http://cibioltg.bitbucket.org.

Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.

DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers.

Characterization and standardization of inducible transcriptional regulators has transformed how scientists approach biology by allowing precise and tunable control of gene expression. Despite their utility, only a handful of well-characterized regulators exist, limiting the complexity of engineered biological systems. We apply a characterization pipeline to four genetically encoded sensors that respond to acrylate, glucarate, erythromycin and naringenin. We evaluate how the concentration of the inducing chemical relates to protein expression, how the extent of induction affects protein expression kinetics, and how the activation behavior of single cells relates to ensemble measurements. We show that activation of each sensor is orthogonal to the other sensors, and to other common inducible systems. We demonstrate independent control of three fluorescent proteins in a single cell, chemically defining eight unique transcriptional states. To demonstrate biosensor utility in metabolic engineering, we apply the glucarate biosensor to monitor product formation in a heterologous glucarate biosynthesis pathway and identify superior enzyme variants. Doubling the number of well-characterized inducible systems makes more complex synthetic biological circuits accessible. Characterizing sensors that transduce the intracellular concentration of valuable metabolites into fluorescent readouts enables high-throughput screening of biological catalysts and alleviates the primary bottleneck of the metabolic engineering design-build-test cycle.