Showing papers in "Nature Structural & Molecular Biology in 2014"
TL;DR: It is shown that a poorly characterized methyltransferase, CMT2, is a functional methyl transferase in vitro and in vivo and suggest that non-CG methylation patterns are critical in shaping the landscapes of histone modification and small noncoding RNA.
Abstract: DNA methylation occurs in CG and non-CG sequence contexts. Non-CG methylation is abundant in plants and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however, its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 preferentially binds histone H3 Lys9 (H3K9) dimethylation and methylates non-CG cytosines that are regulated by H3K9 methylation. We revealed the contributions and redundancies between each non-CG methyltransferase in DNA methylation patterning and in regulating transcription. We also demonstrate extensive dependencies of small-RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the landscapes of histone modification and small noncoding RNA.
670 citations
TL;DR: One lncRNA, Firre, is described that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome, suggesting a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.
Abstract: RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.
562 citations
TL;DR: E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine.
Abstract: E3 ligases carry out the final step in the ubiquitination cascade, catalyzing transfer of ubiquitin from an E2 enzyme to form a covalent bond with a substrate lysine. Three distinct classes of E3 ligases have been identified that stimulate transfer of ubiquitin and ubiquitin-like proteins through either a direct or an indirect mechanism. Only recently have the catalytic mechanisms of E3 ligases begun to be elucidated.
531 citations
TL;DR: This study demonstrates a primary role in DSB repair of the chromatin context in which a break occurs and identifies an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair.
Abstract: Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
515 citations
TL;DR: This work quantitatively studied SUMOylation dynamics in response to SUMO protease inhibition, proteasome inhibition and heat shock, and identified 70 phosphorylation and four acetylation events in proximity to SUMoylation sites, and provides evidence for acetylated-dependent SUMOolation of endogenous histone H3.
Abstract: SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution mass spectrometry, we have studied global SUMOylation in human cells and in a site-specific manner, identifying a total of over 4,300 SUMOylation sites in over 1,600 proteins. Moreover, for the first time in excess of 1,000 SUMOylation sites were identified under standard growth conditions. SUMOylation dynamics were quantitatively studied in response to SUMO protease inhibition, proteasome inhibition and heat shock. A considerable amount of SUMOylated lysines have previously been reported to be ubiquitylated, acetylated or methylated, indicating crosstalk between SUMO and other post-translational modifications. We identified 70 phosphorylation and 4 acetylation events in close proximity to SUMOylation sites, and provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, pre-mRNA splicing and ribosome assembly.
413 citations
TL;DR: It is shown that monoubiquitination of histone H2A by PRC1-type complexes to form H 2Aub creates a binding site for Jarid2–Aebp2–containing PRC2 and promotes H3K27 trimethylation on H2 aub nucleosomes, which constitute components of a positive feedback loop establishing H 3K27me3 chromatin domains.
Abstract: PRC2 promotes methylation of H3K27, a modification that recruits PRC1, which in turn deposits H2A ubiquitin marks. Muller and colleagues use biochemistry approaches to show that H2Aub recruits Jarid–Aebp2–containing PRC2 to promote H3K27 trimethylation on H2Aub nucleosomes, thus forming a positive feedback loop to establish repressed chromatin domains.
387 citations
TL;DR: On the basis of structural comparisons and phylogenetic analyses of pAgos and eAgos, this work reconstructs the evolutionary journey of the Argonaute proteins through the three domains of life and discusses how different structural features of p Agos andeAgos relate to their distinct physiological roles.
Abstract: Argonaute proteins are conserved throughout all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participate in host defense by DNA interference, whereas eukaryotic Argonaute proteins (eAgos) control a wide range of processes by RNA interference. Here we review molecular mechanisms of guide and target binding by Argonaute proteins, and describe how the conformational changes induced by target binding lead to target cleavage. On the basis of structural comparisons and phylogenetic analyses of pAgos and eAgos, we reconstruct the evolutionary journey of the Argonaute proteins through the three domains of life and discuss how different structural features of pAgos and eAgos relate to their distinct physiological roles.
382 citations
TL;DR: It is shown that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determined the 2.3-Å-resolution crystal structure of the Cas1–Cas2 complex, suggesting a mechanism by which Cas1-2 complexes specify sites of CRISPR spacer integration.
Abstract: The initial stage of CRISPR–Cas immunity involves the acquisition of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved amongst all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-A resolution crystal structure of the Cas1–Cas2 complex. Mutations that perturb Cas1–Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Unlike Cas1, active site mutants of Cas2 can still acquire new spacers indicating a non-enzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1–Cas2 complexes specify sites of CRISPR spacer integration.
379 citations
TL;DR: The crystal structure of human CRBN bound to DDB1 and the drug lenalidomide is presented and it is shown that a hydrophobic pocket in the thalidomid-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidmide, whereas the isoindolinone ring is exposed to solvent.
Abstract: The protein Cereblon, part of an ubiquitin E3 ligase complex, is the target for anticancer thalidomide analogs. The crystal structure of human Cereblon-DDB1 with bound lenalidomide reveals how the drug affects E3 substrate recruitment.
363 citations
TL;DR: It is reported that the long terminal repeats of HERVH function as enhancers and that HerVH is a nuclear long noncoding RNA required to maintain hESC identity.
Abstract: Human endogenous retrovirus subfamily H (HERVH) is a class of transposable elements expressed preferentially in human embryonic stem cells (hESCs). Here, we report that the long terminal repeats of HERVH function as enhancers and that HERVH is a nuclear long noncoding RNA required to maintain hESC identity. Furthermore, HERVH is associated with OCT4, coactivators and Mediator subunits. Together, these results uncover a new role of species-specific transposable elements in hESCs.
340 citations
TL;DR: The eukaryotic endoplasmic reticulum (ER) maintains protein homeostasis by eliminating unwanted proteins through the evolutionarily conserved ER-associated degradation (ERAD) pathway and the ubiquitin system is functionally intertwined with retrotranslocation machinery to transport polypeptides across the ER membrane.
Abstract: The endoplasmic reticulum–associated degradation (ERAD) pathway maintains protein homeostasis in the ER by retrotranslocating unwanted proteins to the cytosol for proteasomal degradation. This Review discusses the integral role of the ubiquitin system in ERAD, highlighting how the two pathways intertwine to facilitate transport across the ER membrane. The eukaryotic endoplasmic reticulum (ER) maintains protein homeostasis by eliminating unwanted proteins through the evolutionarily conserved ER-associated degradation (ERAD) pathway. During ERAD, maturation-defective and surplus polypeptides are evicted from the ER lumen and/or lipid bilayer through the process of retrotranslocation and ultimately degraded by the proteasome. An integral facet of the ERAD mechanism is the ubiquitin system, composed of the ubiquitin modifier and the factors for assembling, processing and binding ubiquitin chains on conjugated substrates. Beyond simply marking polypeptides for degradation, the ubiquitin system is functionally intertwined with retrotranslocation machinery to transport polypeptides across the ER membrane.
TL;DR: The cocrystal structure of Spinach bound to its cognate exogenous chromophore is solved, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G.
Abstract: GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
TL;DR: Genome-wide analyses have revealed chromatin signatures of enhancers, and enhancer signatures have been used to describe the transitions of these regulatory elements from inactive to primed and from activated to decommissioned states during development.
Abstract: Enhancers are cis-regulatory elements that enable precise spatiotemporal patterns of gene expression during development and are notable for being able to function at large distances from their target genes. Such regulatory elements often bypass intervening genes and typically comprise binding sites for multiple transcription factors that can also be transcribed by RNA polymerase II (Pol II) to produce noncoding enhancer RNAs (eRNAs). Genome-wide analyses have revealed chromatin signatures of enhancers, such as the enrichment for monomethylation of histone H3 lysine 4 (H3K4me1) and the acetylation or methylation of histone H3 lysine 27 (H3K27). Enhancer signatures have been used to describe the transitions of these regulatory elements from inactive to primed and from activated to decommissioned states during development. New mutations of enhancer sequences and of the protein factors regulating enhancer function in human disease continue to be identified, contributing to a growing class of 'enhanceropathies'.
TL;DR: The structures reveal that c-di-GMP releases an autoinhibited state of the enzyme by breaking a salt bridge that otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site.
Abstract: The bacterial signaling molecule cyclic di-GMP (c-di-GMP) stimulates the synthesis of bacterial cellulose, which is frequently found in biofilms. Bacterial cellulose is synthesized and translocated across the inner membrane by a complex of cellulose synthase BcsA and BcsB subunits. Here we present crystal structures of the c-di-GMP-activated BcsA-BcsB complex. The structures reveal that c-di-GMP releases an autoinhibited state of the enzyme by breaking a salt bridge that otherwise tethers a conserved gating loop that controls access to and substrate coordination at the active site. Disrupting the salt bridge by mutagenesis generates a constitutively active cellulose synthase. Additionally, the c-di-GMP-activated BcsA-BcsB complex contains a nascent cellulose polymer whose terminal glucose unit rests at a new location above BcsA's active site and is positioned for catalysis. Our mechanistic insights indicate how c-di-GMP allosterically modulates enzymatic functions.
TL;DR: It is shown that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function.
Abstract: Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function. BRD4 interacts with elongating Pol II complexes and assists Pol II in progression through hyperacetylated nucleosomes by interacting with acetylated histones via bromodomains. On active enhancers, the BET inhibitor JQ1 antagonizes BRD4-associated eRNA synthesis. Thus, BRD4 is involved in multiple steps of the transcription hierarchy, primarily by facilitating transcript elongation both at enhancers and on gene bodies independently of P-TEFb.
TL;DR: Structural mechanisms underlying the functions and conjugation of a pair of distinctive ubiquitin-like proteins, Atg8 and Atg12, that are specialized to provide interaction platforms linked to phagophore membranes are reviewed.
Abstract: Whereas the proteasome degrades individual proteins modified with ubiquitin chains, autophagy degrades many proteins and organelles en masse. A pair of ubiquitin-like proteins (UBLs), Atg8 and Atg12, regulate autophagy-mediated degradation in a manner completely distinct from that of ubiquitin in the proteasome pathway, as discussed in this Review.
TL;DR: The induction of ALT phenotypes as a consequence of ASF1 depletion strongly supports the hypothesis that ALT is a result of histone management dysfunction.
Abstract: The mechanism of activation of the Alternative Lengthening of Telomeres (ALT) pathway of mammalian chromosome end maintenance has remained an unresolved issue. We have discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT associated PML bodies (APBs), extra-chromosomal telomeric DNA species an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and inter-telomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We identified that ALT induction is positively regulated by RAD17 and BLM, while negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly support the hypothesis that ALT is a consequence of a histone management dysfunction.
TL;DR: It is proposed that cotranslational regulation of nascent-chain fate may be a general constraint shaping codon usage in the genome and modulation of local elongation rates through codon choice appears to kinetically enhance recognition by ribosome-associated factors.
Abstract: Analyses of yeast codon usage and ribosome profiling data reveal a nonoptimal codon cluster in the mRNAs of ER-targeted proteins, downstream of the SRP-binding site, that would slow down translation to promote SRP interaction.
TL;DR: In vivo decay assays indicate that this blunt-ended triple helix, with the 3′ nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay, which explains why triple-helical stacks longer than six do not occur in nature.
Abstract: The X-ray crystal structure and biochemical analysis of a triple helix formed between the expression and nuclear retention element (ENE) and the 3′ poly(A) tail of the human long noncoding RNA MALAT-1 reveals the basis of its stability and how it confers resistance to degradation.
TL;DR: It is proposed that, unlike earlier models, the ribosome and the A-site tRNA facilitate the deprotonation of the nucleophile through the activation of a water molecule.
Abstract: During peptide-bond formation on the ribosome, the α-amine of an aminoacyl-tRNA attacks the ester carbonyl carbon of a peptidyl-tRNA to yield a peptide lengthened by one amino acid. Although the ribosome's contribution to catalysis is predominantly entropic, the lack of high-resolution structural data for the complete active site in complex with full-length ligands has made it difficult to assess how the ribosome might influence the pathway of the reaction. Here, we present crystal structures of preattack and postcatalysis complexes of the Thermus thermophilus 70S ribosome at ~2.6-A resolution. These structures reveal a network of hydrogen bonds along which proton transfer could take place to ensure the concerted, rate-limiting formation of a tetrahedral intermediate. We propose that, unlike earlier models, the ribosome and the A-site tRNA facilitate the deprotonation of the nucleophile through the activation of a water molecule.
TL;DR: A method is described for high-resolution mapping of underwound DNA, using next-generation sequencing, and torsion is correlated with gene expression in Drosophila melanogaster cells, suggesting that the elongation-independent effects of torsional stress on nucleosome dynamics contributes to the destabilization of nucleosomes.
Abstract: As RNA polymerase II (Pol II) transcribes a gene, it encounters an array of well-ordered nucleosomes. How it traverses through this array in vivo remains unresolved. One model proposes that torsional stress generated during transcription destabilizes nucleosomes ahead of Pol II. Here, we describe a method for high-resolution mapping of underwound DNA, using next-generation sequencing, and show that torsion is correlated with gene expression in Drosophila melanogaster cells. Accumulation of torsional stress, through topoisomerase inhibition, results in increased Pol II at transcription start sites. Whereas topoisomerase I inhibition results in increased nascent RNA transcripts, topoisomerase II inhibition causes little change. Despite the different effects on Pol II elongation, topoisomerase inhibition results in increased nucleosome turnover and salt solubility within gene bodies, thus suggesting that the elongation-independent effects of torsional stress on nucleosome dynamics contributes to the destabilization of nucleosomes.
TL;DR: Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain, and reconstituted Saccharomyces cerevisiae replication forks found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand.
Abstract: Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.
TL;DR: It is proposed that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.
Abstract: Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.
TL;DR: The crystal structure of Staphylococcus capitis DMT, a close prokaryotic homolog of the SLC11 family, is determined and reveals a conserved mechanism for transition-metal ion selectivity within the S LC11 family.
Abstract: The X-ray crystal structure of ScaDMT, a bacterial member of the solute carrier 11 transporter family, identifies conserved residues within the substrate-binding site that confer metal-ion selectivity.
TL;DR: Dephosphorylation of specific serines in Atg13 enhanced its interaction with not only Atg1 but also Atg17, and this promotes PAS assembly and autophagy progression.
Abstract: Autophagy initiates with the assembly of a preautophagosomal structure (PAS), triggered by the yeast Atg1 complex. Ohsumi, Noda and colleagues present the crystal structures of Atg13–Atg1 and Atg13–Atg17, revealing how starvation-induced dephosphorylation of Atg13 triggers formationof the Atg1 complex and PAS assembly.
TL;DR: The T-cell receptor (TCR) usage and fine specificity of patient-derived T- cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and D Q2.
Abstract: Celiac disease is a T cell-mediated disease induced by dietary gluten, a component of which is gliadin. 95% of individuals with celiac disease carry the HLA (human leukocyte antigen)-DQ2 locus. Here we determined the T-cell receptor (TCR) usage and fine specificity of patient-derived T-cell clones specific for two epitopes from wheat gliadin, DQ2.5-glia-α1a and DQ2.5-glia-α2. We determined the ternary structures of four distinct biased TCRs specific for those epitopes. All three TCRs specific for DQ2.5-glia-α2 docked centrally above HLA-DQ2, which together with mutagenesis and affinity measurements provided a basis for the biased TCR usage. A non-germline encoded arginine residue within the CDR3β loop acted as the lynchpin within this common docking footprint. Although the TCRs specific for DQ2.5-glia-α1a and DQ2.5-glia-α2 docked similarly, their interactions with the respective gliadin determinants differed markedly, thereby providing a basis for epitope specificity.
TL;DR: Cryo-EM was used to define the secretion path, providing a structural explanation as to why effector proteins must be unfolded during transport and showing that mechanisms rejecting unacceptable substrates can be undermined, and transport of bacterial effectors across an already assembled type 3 secretion system can be inhibited.
Abstract: The type III secretion systems of infectious bacteria use the needle-like injectisome to secrete proteins from the bacterial cytoplasm into host cells. Cryo-electron tomography and single-particle cryo-EM reveal for the first time the path of unfolded protein substrates through the narrow bore of the injectisome.
TL;DR: A crystal structure of mouse Hv1 in the resting state showing a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule provides a platform for understanding the general principles of voltage sensing and proton permeation.
Abstract: The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-A resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.
TL;DR: The crystal structures of Thermobifida fusca Cas3 bound to single-stranded DNA substrate are reported and it is shown that it is an obligate 3′-to-5′ ssDNase that preferentially accepts substrate directly from the helicase moiety.
Abstract: Type I CRISPR-Cas systems require a target-searching Cascade complex and the Cas3 degradation machine to drive prokaryotic adaptation to alien nucleic acids. Cas3 crystal structures now reveal the mechanism of concerted DNA unwinding and degradation.
TL;DR: It is reported that U2AF has the capacity to directly define ~88% of functional 3′ splice sites in the human genome, but numerous U2 AF binding events also occur in intronic locations.
Abstract: The U2AF heterodimer has been well studied for its role in defining functional 3' splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3' splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3' splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states