FANCJ is essential to maintain microsatellite structure genome-wide during replication stress
(Source: Nucleic Acids Research)
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Barthelemy, J., Hanenberg, H., Leffak, M. Tags: Corrigendum Source Type: research
A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae
Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via ...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Reider Apel, A., d'Espaux, L., Wehrs, M., Sachs, D., Li, R. A., Tong, G. J., Garber, M., Nnadi, O., Zhuang, W., Hillson, N. J., Keasling, J. D., Mukhopadhyay, A. Tags: Synthetic Biology and Assembly Cloning Synthetic Biology and Bioengineering Source Type: research
Structure and folding of the Tetrahymena telomerase RNA pseudoknot
Telomerase maintains telomere length at the ends of linear chromosomes using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). An essential part of TER is the template/pseudoknot domain (t/PK) which includes the template, for adding telomeric repeats, template boundary element (TBE), and pseudoknot, enclosed in a circle by stem 1. The Tetrahymena telomerase holoenzyme catalytic core (p65-TER-TERT) was recently modeled in our 9 Å resolution cryo-electron microscopy map by fitting protein and TER domains, including a solution NMR structure of the Tetrahymena pseudoknot. Here, we describe in ...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Cash, D. D., Feigon, J. Tags: Structural Biology Source Type: research
Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction
Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SR...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Becker, M. M. M., Lapouge, K., Segnitz, B., Wild, K., Sinning, I. Tags: Structural Biology Source Type: research
Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers
Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Pica, A., Russo Krauss, I., Parente, V., Tateishi-Karimata, H., Nagatoishi, S., Tsumoto, K., Sugimoto, N., Sica, F. Tags: Structural Biology Source Type: research
Thermotoga maritima NusG: domain interaction mediates autoinhibition and thermostability
NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is in...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Drögemüller, J., Schneider, C., Schweimer, K., Strauss, M., Wöhrl, B. M., Rösch, P., Knauer, S. H. Tags: Structural Biology Source Type: research
Reactive sulfur species regulate tRNA methylthiolation and contribute to insulin secretion
The 2-methylthio (ms2) modification at A37 of tRNAs is critical for accurate decoding, and contributes to metabolic homeostasis in mammals. However, the regulatory mechanism of ms2 modification remains largely unknown. Here, we report that cysteine hydropersulfide (CysSSH), a newly identified reactive sulfur species, is involved in ms2 modification in cells. The suppression of intracellular CysSSH production rapidly reduced ms2 modification, which was rescued by the application of an exogenous CysSSH donor. Using a unique and stable isotope-labeled CysSSH donor, we show that CysSSH was capable of specifically transferring ...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Takahashi, N., Wei, F.-Y., Watanabe, S., Hirayama, M., Ohuchi, Y., Fujimura, A., Kaitsuka, T., Ishii, I., Sawa, T., Nakayama, H., Akaike, T., Tomizawa, K. Tags: RNA Source Type: research
Alternative splicing of U2AF1 reveals a shared repression mechanism for duplicated exons
The auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation of lariat introns. The gene for the 35-kD subunit of U2AF gives rise to two protein isoforms (termed U2AF35a and U2AF35b) that are encoded by alternatively spliced exons 3 and Ab, respectively. The splicing recognition sequences of exon 3 are less favorable than exon Ab, yet U2AF35a expression is higher than U2AF35b across tissues. We show that U2AF35b repression is facilitated by weak, closely spaced BPs next to a long polypyrimidine tract of exon Ab. Each BP lacked canonical uridines at position -2 re...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Kralovicova, J., Vorechovsky, I. Tags: RNA Source Type: research
RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns
We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show elevated U12-intron retention in all examined tissues from SMA mice, and that U12-dependent intron retention is induced upon siRNA knock-down of SMN in HeLa cells. Furthermore, we show that retention of U12-dependent introns is mitigated by ASO treatment of SMA mice and that many transcriptional changes are reversed. Finally, we report on missplicing of several Ca2+ channel genes that may explain disrupted Ca2+ homeostasis in SMA and activation of Cdk5. (Source: Nucleic Acids Research)
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Doktor, T. K., Hua, Y., Andersen, H. S., Broner, S., Liu, Y. H., Wieckowska, A., Dembic, M., Bruun, G. H., Krainer, A. R., Andresen, B. S. Tags: RNA Source Type: research
The bromodomain protein BRD4 regulates splicing during heat shock
The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Hussong, M., Kaehler, C., Kerick, M., Grimm, C., Franz, A., Timmermann, B., Welzel, F., Isensee, J., Hucho, T., Krobitsch, S., Schweiger, M. R. Tags: RNA Source Type: research
Asymmetric positioning of Cas1-2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system
CRISPR–Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Yoganand, K. N. R., Sivathanu, R., Nimkar, S., Anand, B. Tags: Nucleic Acid Enzymes Source Type: research
Temporal self-regulation of transposition through host-independent transposase rodlet formation
Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet f...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Woodard, L. E., Downes, L. M., Lee, Y.-C., Kaja, A., Terefe, E. S., Wilson, M. H. Tags: Nucleic Acid Enzymes Source Type: research
Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions
RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to ...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Konomura, N., Arai, N., Shinohara, T., Kobayashi, J., Iwasaki, W., Ikawa, S., Kusano, K., Shibata, T. Tags: Nucleic Acid Enzymes Source Type: research
A ribosome profiling study of mRNA cleavage by the endonuclease RelE
Implicated in persistence and stress response pathways in bacteria, RelE shuts down protein synthesis by cleaving mRNA within the ribosomal A site. Structural and biochemical studies have shown that RelE cuts with some sequence specificity, which we further characterize here, and that it shows no activity outside the context of the ribosome. We obtained a global view of the effect of RelE on translation by ribosome profiling, observing that ribosomes accumulate on the 5'-end of genes through dynamic cycles of mRNA cleavage, ribosome rescue and initiation. Moreover, the addition of purified RelE to cell lysates shows promis...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Hwang, J.-Y., Buskirk, A. R. Tags: Nucleic Acid Enzymes Source Type: research
Regulated complex assembly safeguards the fidelity of Sleeping Beauty transposition
The functional relevance of the inverted repeat structure (IR/DR) in a subgroup of the Tc1/mariner superfamily of transposons has been enigmatic. In contrast to mariner transposition, where a topological filter suppresses single-ended reactions, the IR/DR orchestrates a regulatory mechanism to enforce synapsis of the transposon ends before cleavage by the transposase occurs. This ordered assembly process shepherds primary transposase binding to the inner 12DRs (where cleavage does not occur), followed by capture of the 12DR of the other transposon end. This extra layer of regulation suppresses aberrant, potentially genotox...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Wang, Y., Pryputniewicz-Dobrinska, D., Nagy, E. E., Kaufman, C. D., Singh, M., Yant, S., Wang, J., Dalda, A., Kay, M. A., Ivics, Z., Izsvak, Z. Tags: Molecular Biology Source Type: research
