Nature Structural & Molecular Biology

Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5.

Publication Date: 2013 Jun 9 PMID: 23748380
Authors: Kassube, S. A. - Jinek, M. - Fang, J. - Tsutakawa, S. - Nogales, E.
Journal: Nat Struct Mol Biol

RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-EM structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction.

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A bacterial-like mechanism for transcription termination by the Sen1p helicase in budding yeast.

Publication Date: 2013 Jun 9 PMID: 23748379
Authors: Porrua, O. - Libri, D.
Journal: Nat Struct Mol Biol

Transcription termination is essential to generate functional RNAs and to prevent disruptive polymerase collisions resulting from concurrent transcription. The yeast Sen1p helicase is involved in termination of most noncoding RNAs transcribed by RNA polymerase II (RNAPII). However, the mechanism of termination and the role of this protein have remained enigmatic. Here we address the mechanism of Sen1p-dependent termination by using a highly purified in vitro system. We show that Sen1p is the key enzyme of the termination reaction and reveal features of the termination mechanism. Like the bacterial termination factor Rho, Sen1p recognizes the nascent RNA and hydrolyzes ATP to dissociate the elongation complex. Sen1p-dependent termination is highly specific and, notably, does not require the C-terminal domain of RNAPII. We also show that termination is inhibited by RNA-DNA hybrids. Our results elucidate the role of Sen1p in controlling pervasive transcription.

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Molecular dissection of human Argonaute proteins by DNA shuffling.

Publication Date: 2013 Jun 9 PMID: 23748378
Authors: Schurmann, N. - Trabuco, L. G. - Bender, C. - Russell, R. B. - Grimm, D.
Journal: Nat Struct Mol Biol

A paramount task in RNA interference research is to decipher the complex biology of cellular effectors, exemplified in humans by four pleiotropic Argonaute proteins (Ago1-Ago4). Here, we exploited DNA family shuffling, a molecular evolution technology, to generate chimeric Ago protein libraries for dissection of intricate phenotypes independently of prior structural knowledge. Through shuffling of human Ago2 and Ago3, we discovered two N-terminal motifs that govern RNA cleavage in concert with the PIWI domain. Structural modeling predicts an impact on protein rigidity and/or RNA-PIWI alignment, suggesting new mechanistic explanations for Ago3's slicing deficiency. Characterization of hybrids including Ago1 and Ago4 solidifies that slicing requires the juxtaposition and combined action of multiple disseminated modules. We also present a Gateway library of codon-optimized chimeras of human Ago1-Ago4 and molecular evolution analysis software as resources for future investigations into RNA interference sequence-structure-function relationships.

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Scratching the (lateral) surface of chromatin regulation by histone modifications.

Publication Date: 2013 Jun PMID: 23739170
Authors: Tropberger, P. - Schneider, R.
Journal: Nat Struct Mol Biol

Histones have two structurally and functionally distinct domains: globular domains forming the nucleosomal core around which DNA is wrapped and unstructured tails protruding from the nucleosomal core. Whereas post-translational modifications (PTMs) in histone tails are well studied, much less is currently known about histone-core PTMs. Many core PTMs map to residues located on the lateral surface of the histone octamer, close to the DNA, and they have the potential to alter intranucleosomal histone-DNA interactions. Here we discuss recent advances in understanding the function of lateral-surface PTMs. Whereas modifications in the histone tails might have limited structural impact on the nucleosome itself and function as signals to recruit specific binding proteins, PTMs in the lateral surface can have a direct structural effect on nucleosome and chromatin dynamics, even in the absence of specific binding proteins, which adds a twist to the debate on the functionality and causality of PTMs.

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Getting your vitamins-out.

Publication Date: 2013 Jun PMID: 23739169
Authors: Chen, I.
Journal: Nat Struct Mol Biol

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mTOR: restricted access.

Publication Date: 2013 Jun PMID: 23739168
Authors: Larochelle, S.
Journal: Nat Struct Mol Biol

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This is about the in and the out.

Publication Date: 2013 Jun PMID: 23739167
Authors: Henderson, P. J. - Baldwin, S. A.
Journal: Nat Struct Mol Biol

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Making sense of nonsense.

Publication Date: 2013 Jun PMID: 23739166
Authors: Hentze, M. W. - Izaurralde, E.
Journal: Nat Struct Mol Biol

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Solo or doppio: how many CENP-As make a centromeric nucleosome?

Publication Date: 2013 Jun PMID: 23739165
Authors: Dunleavy, E. M. - Zhang, W. - Karpen, G. H.
Journal: Nat Struct Mol Biol

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Toggling in the spliceosome.

Publication Date: 2013 Jun PMID: 23739164
Authors: Abelson, J.
Journal: Nat Struct Mol Biol

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A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression.

Publication Date: 2013 Jun 2 PMID: 23728293
Authors: Winkler, A. - Heintz, U. - Lindner, R. - Reinstein, J. - Shoeman, R. L. - Schlichting, I.
Journal: Nat Struct Mol Biol

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.

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The glucocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals.

Publication Date: 2013 Jun 2 PMID: 23728292
Authors: Watson, L. C. - Kuchenbecker, K. M. - Schiller, B. J. - Gross, J. D. - Pufall, M. A. - Yamamoto, K. R.
Journal: Nat Struct Mol Biol

Glucocorticoid receptor (GR) binds to genomic response elements and regulates gene transcription with cell and gene specificity. Within a response element, the precise sequence to which the receptor binds has been implicated in directing its structure and activity. Here, we use NMR chemical-shift difference mapping to show that nonspecific interactions with bases at particular positions in the binding sequence, such as those of the 'spacer', affect the conformation of distinct regions of the rat GR DNA-binding domain. These regions include the DNA-binding surface, the 'lever arm' and the dimerization interface, suggesting an allosteric pathway that signals between the DNA-binding sequence and the associated dimer partner. Disrupting this pathway by mutating the dimer interface alters sequence-specific conformations, DNA-binding kinetics and transcriptional activity. Our study demonstrates that GR dimer partners collaborate to read DNA shape and to direct sequence-specific gene activity.

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Nucleosome dynamics regulates DNA processing.

Publication Date: 2013 Jun 2 PMID: 23728291
Authors: Adkins, N. L. - Niu, H. - Sung, P. - Peterson, C. L.
Journal: Nat Struct Mol Biol

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. The first step in DSB repair by homologous recombination is the processing of the ends by one of two resection pathways, executed by the Saccharomyces cerevisiae Exo1 and Sgs1-Dna2 machineries. Here we report in vitro and in vivo studies that characterize the impact of chromatin on each resection pathway. We find that efficient resection by the Sgs1-Dna2-dependent machinery requires a nucleosome-free gap adjacent to the DSB. Resection by Exo1 is blocked by nucleosomes, and processing activity can be partially restored by removal of the H2A-H2B dimers. Our study also supports a role for the dynamic incorporation of the H2A.Z histone variant in Exo1 processing, and it further suggests that the two resection pathways require distinct chromatin remodeling events to navigate chromatin structure.

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Integrative genomic analyses reveal clinically relevant long noncoding RNAs in human cancer.

Publication Date: 2013 Jun 2 PMID: 23728290
Authors: Du, Z. - Fei, T. - Verhaak, R. G. - Su, Z. - Zhang, Y. - Brown, M. - Chen, Y. - Liu, X. S.
Journal: Nat Struct Mol Biol

Despite growing appreciation of the importance of long noncoding RNAs (lncRNAs) in normal physiology and disease, our knowledge of cancer-related lncRNAs remains limited. By repurposing microarray probes, we constructed expression profiles of 10,207 lncRNA genes in approximately 1,300 tumors over four different cancer types. Through integrative analysis of the lncRNA expression profiles with clinical outcome and somatic copy-number alterations, we identified lncRNAs that are associated with cancer subtypes and clinical prognosis and predicted those that are potential drivers of cancer progression. We validated our predictions by experimentally confirming prostate cancer cell growth dependence on two newly identified lncRNAs. Our analysis provides a resource of clinically relevant lncRNAs for the development of lncRNA biomarkers and the identification of lncRNA therapeutic targets. It also demonstrates the power of integrating publically available genomic data sets and clinical information for discovering disease-associated lncRNAs.

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Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.

Publication Date: 2013 May 26 PMID: 23708608
Authors: Qi, R. - Sarbeng, E. B. - Liu, Q. - Le, K. Q. - Xu, X. - Xu, H. - Yang, J. - Wong, J. L. - Vorvis, C. - Hendrickson, W. A. - Zhou, L. - Liu, Q.
Journal: Nat Struct Mol Biol

The 70-kilodalton (kDa) heat-shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD), which binds and hydrolyzes ATP, and a substrate-binding domain (SBD), which binds extended polypeptides. NBD and SBD interact little when in the presence of ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact ATP-bound Hsp70 from Escherichia coli at 1.96-A resolution. The ATP-bound NBD adopts a unique conformation, forming extensive interfaces with an SBD that has changed radically, having its alpha-helical lid displaced and the polypeptide-binding channel of its beta-subdomain restructured. These conformational changes, together with our biochemical assays, provide a structural explanation for allosteric coupling in Hsp70 activity.

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Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16.

Publication Date: 2013 May 26 PMID: 23708607
Authors: Pancera, M. - Shahzad-Ul-Hussan, S. - Doria-Rose, N. A. - McLellan, J. S. - Bailer, R. T. - Dai, K. - Loesgen, S. - Louder, M. K. - Staupe, R. P. - Yang, Y. - Zhang, B. - Parks, R. - Eudailey, J. - Lloyd, K. E. - Blinn, J. - Alam, S. M. - Haynes, B. F. - Amin, M. N. - Wang, L. X. - Burton, D. R. - Koff, W. C. - Nabel, G. J. - Mascola, J. R. - Bewley, C. A. - Kwong, P. D.
Journal: Nat Struct Mol Biol

HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1-V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1-V2, showing an epitope comprising both high mannose-type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1-glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

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Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120.

Publication Date: 2013 May 26 PMID: 23708606
Authors: Kong, L. - Lee, J. H. - Doores, K. J. - Murin, C. D. - Julien, J. P. - McBride, R. - Liu, Y. - Marozsan, A. - Cupo, A. - Klasse, P. J. - Hoffenberg, S. - Caulfield, M. - King, C. R. - Hua, Y. - Le, K. M. - Khayat, R. - Deller, M. C. - Clayton, T. - Tien, H. - Feizi, T. - Sanders, R. W. - Paulson, J. C. - Moore, J. P. - Stanfield, R. L. - Burton, D. R. - Ward, A. B. - Wilson, I. A.
Journal: Nat Struct Mol Biol

A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan-dependent epitope from its 3.1-A crystal structure with gp120, CD4 and Fab 17b. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield and access the gp120 protein surface. EM reveals that PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. Combined structural studies of PGT 135, PGT 128 and 2G12 show that this Asn332-dependent antigenic region is highly accessible and much more extensive than initially appreciated, which allows for multiple binding modes and varied angles of approach; thereby it represents a supersite of vulnerability for antibody neutralization.

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Electron microscopy structure of human APC/C-EMI1 reveals multimodal mechanism of E3 ligase shutdown.

Publication Date: 2013 May 26 PMID: 23708605
Authors: Frye, J. J. - Brown, N. G. - Petzold, G. - Watson, E. R. - Grace, C. R. - Nourse, A. - Jarvis, M. A. - Kriwacki, R. W. - Peters, J. M. - Stark, H. - Schulman, B. A.
Journal: Nat Struct Mol Biol

The anaphase-promoting complex/cyclosome (APC/C) is a ~1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/CCDH1 during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/CCDH1 functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/CCDH1 to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.

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Homeostatic control of Argonaute stability by microRNA availability.

Publication Date: 2013 May 26 PMID: 23708604
Authors: Smibert, P. - Yang, J. S. - Azzam, G. - Liu, J. L. - Lai, E. C.
Journal: Nat Struct Mol Biol

Homeostatic mechanisms regulate the abundance of several components in small-RNA pathways. We used Drosophila and mammalian systems to demonstrate a conserved homeostatic system in which the status of miRNA biogenesis controls Argonaute protein stability. Clonal analyses of multiple mutants of core Drosophila miRNA factors revealed that stability of the miRNA effector AGO1 is dependent on miRNA biogenesis. Reciprocally, ectopic transcription of miRNAs within in vivo clones induced accumulation of AGO1, as did genetic interference with the ubiquitin-proteasome system. In mouse cells, we found that the stability of Ago2 declined in Dicer-knockout cells and was rescued by proteasome blockade or introduction of either Dicer plasmid or Dicer-independent miRNA constructs. Notably, Dicer-dependent miRNA constructs generated pre-miRNAs that bound Ago2 but did not rescue Ago2 stability. We conclude that Argonaute levels are finely tuned by cellular availability of mature miRNAs and the ubiquitin-proteasome system.

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Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.

Publication Date: 2013 Jun PMID: 23686288
Authors: Pomerantz, R. T. - Kurth, I. - Goodman, M. F. - O'Donnell, M. E.
Journal: Nat Struct Mol Biol

Although homologous recombination is considered an accurate form of DNA repair, genetics suggest that the Escherichia coli translesion DNA polymerase IV (Pol IV, also known as DinB) promotes error-prone recombination during stress, which allows cells to overcome adverse conditions. However, how Pol IV functions and is regulated during recombination under stress is unknown. We show that Pol IV is highly proficient in error-prone recombination and is preferentially recruited to displacement loops (D loops) at stress-induced concentrations in vitro. We also found that high-fidelity Pol II switches to exonuclease mode at D loops, which is stimulated by topological stress and reduced deoxyribonucleotide pool concentration during stationary phase. The exonuclease activity of Pol II enables it to compete with Pol IV, which probably suppresses error-prone recombination. These findings indicate that preferential D-loop extension by Pol IV facilitates error-prone recombination and explain how Pol II reduces such errors in vivo.

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A conformational switch in PRP8 mediates metal ion coordination that promotes pre-mRNA exon ligation.

Publication Date: 2013 Jun PMID: 23686287
Authors: Schellenberg, M. J. - Wu, T. - Ritchie, D. B. - Fica, S. - Staley, J. P. - Atta, K. A. - Lapointe, P. - Macmillan, A. M.
Journal: Nat Struct Mol Biol

Splicing of pre-mRNAs in eukaryotes is catalyzed by the spliceosome, a large RNA-protein metalloenzyme. The catalytic center of the spliceosome involves a structure comprising the U2 and U6 snRNAs and includes a metal bound by U6 snRNA. The precise architecture of the splicesome active site, however, and the question of whether it includes protein components, remains unresolved. A wealth of evidence places the protein PRP8 at the heart of the spliceosome through assembly and catalysis. Here we provide evidence that the RNase H domain of PRP8 undergoes a conformational switch between the two steps of splicing, rationalizing yeast prp8 alleles that promote either the first or second step. We also show that this switch unmasks a metal-binding site involved in the second step. Together, these data establish that PRP8 is a metalloprotein that promotes exon ligation within the spliceosome.

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Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.

Publication Date: 2013 Jun PMID: 23665582
Authors: Honigmann, A. - van den Bogaart, G. - Iraheta, E. - Risselada, H. J. - Milovanovic, D. - Mueller, V. - Mullar, S. - Diederichsen, U. - Fasshauer, D. - Grubmuller, H. - Hell, S. W. - Eggeling, C. - Kuhnel, K. - Jahn, R.
Journal: Nat Struct Mol Biol

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

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eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells.

Publication Date: 2013 Jun PMID: 23665581
Authors: Rufener, S. C. - Muhlemann, O.
Journal: Nat Struct Mol Biol

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 implies a similar basic mechanism of PTC recognition in all eukaryotes. However, while PTC-containing mRNAs in yeast seem to be available to NMD at each round of translation, mammalian NMD has been reported to be restricted to cap-binding complex (CBC)-bound mRNAs during the pioneer round of translation. Here, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and demonstrate that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

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Nonsense-mediated mRNA decay occurs during eIF4F-dependent translation in human cells.

Publication Date: 2013 Jun PMID: 23665580
Authors: Durand, S. - Lykke-Andersen, J.
Journal: Nat Struct Mol Biol

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs undergoing premature termination of translation. It has been argued that in human cells, NMD is restricted to a pioneer round of translation initiated on mRNAs associated with the cap-binding complex (CBC) and that the exchange of the CBC for the eIF4F translation initiation complex renders mRNAs immune to NMD. Here, we demonstrate that human mRNAs undergoing eIF4F-dependent translation are not immune to NMD. First, prolonged translation inhibition does not render an NMD substrate resistant to NMD, despite allowing exchange of CBC for eIF4F. Second, eIF4F inhibitors stabilize NMD substrates undergoing cap-dependent translation. Third, the eIF4E-associated pool of an NMD substrate degrades as rapidly as the overall pool of the mRNA. Thus, eIF4F-dependent translation supports NMD in human cells, allowing for the possibility that NMD could be activated upon cellular cues on already translating mRNAs.

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Telomere position effect regulates DUX4 in human facioscapulohumeral muscular dystrophy.

Publication Date: 2013 Jun PMID: 23644600
Authors: Stadler, G. - Rahimov, F. - King, O. D. - Chen, J. C. - Robin, J. D. - Wagner, K. R. - Shay, J. W. - Emerson, C. P. Jr - Wright, W. E.
Journal: Nat Struct Mol Biol

Telomeres may regulate human disease by at least two independent mechanisms. First, replicative senescence occurs once short telomeres generate DNA-damage signals that produce a barrier to tumor progression. Second, telomere position effects (TPE) could change gene expression at intermediate telomere lengths in cultured human cells. Here we report that telomere length may contribute to the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a late-onset disease genetically residing only 25-60 kilobases from the end of chromosome 4q. We used a floxable telomerase to generate isogenic clones with different telomere lengths from affected patients and their unaffected siblings. DUX4, the primary candidate for FSHD pathogenesis, is upregulated over ten-fold in FSHD myoblasts and myotubes with short telomeres, and its expression is inversely proportional to telomere length. FSHD may be the first known human disease in which TPE contributes to age-related phenotype.

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Structural basis for the recruitment of the human CCR4-NOT deadenylase complex by tristetraprolin.

Publication Date: 2013 Jun PMID: 23644599
Authors: Fabian, M. R. - Frank, F. - Rouya, C. - Siddiqui, N. - Lai, W. S. - Karetnikov, A. - Blackshear, P. J. - Nagar, B. - Sonenberg, N.
Journal: Nat Struct Mol Biol

Tristetraprolin (TTP) is an RNA-binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3' untranslated regions of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multisubunit CCR4-NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds a central domain of CNOT1, a core subunit of the CCR4-NOT complex. A high-resolution crystal structure of the TTP-CNOT1 complex was determined, providing the first structural insight, to our knowledge, into an ARE-binding protein bound to the CCR4-NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.

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CENP-A confers a reduction in height on octameric nucleosomes.

Publication Date: 2013 Jun PMID: 23644598
Authors: Miell, M. D. - Fuller, C. J. - Guse, A. - Barysz, H. M. - Downes, A. - Owen-Hughes, T. - Rappsilber, J. - Straight, A. F. - Allshire, R. C.
Journal: Nat Struct Mol Biol

Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.

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Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming.

Publication Date: 2013 Jun PMID: 23644597
Authors: Maspero, E. - Valentini, E. - Mari, S. - Cecatiello, V. - Soffientini, P. - Pasqualato, S. - Polo, S.
Journal: Nat Struct Mol Biol

Homologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4). The HECT(Nedd4)~Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue.

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The octamer is the major form of CENP-A nucleosomes at human centromeres.

Publication Date: 2013 Jun PMID: 23644596
Authors: Hasson, D. - Panchenko, T. - Salimian, K. J. - Salman, M. U. - Sekulic, N. - Alonso, A. - Warburton, P. E. - Black, B. E.
Journal: Nat Struct Mol Biol

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on alpha-satellite 171-base-pair monomers at normal centromeres and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA-wrapping behavior, as do octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on conventional nucleosomes containing canonical histone H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.

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Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity.

Publication Date: 2013 Jun PMID: 23644595
Authors: Rajagopalan, S. - Teter, S. J. - Zwart, P. H. - Brennan, R. G. - Phillips, K. J. - Kiley, P. J.
Journal: Nat Struct Mol Biol

IscR from Escherichia coli is an unusual metalloregulator in that both apo and iron sulfur (Fe-S)-IscR regulate transcription and exhibit different DNA binding specificities. Here, we report structural and biochemical studies of IscR suggesting that remodeling of the protein-DNA interface upon Fe-S ligation broadens the DNA binding specificity of IscR from binding the type 2 motif only to both type 1 and type 2 motifs. Analysis of an apo-IscR variant with relaxed target-site discrimination identified a key residue in wild-type apo-IscR that, we propose, makes unfavorable interactions with a type 1 motif. Upon Fe-S binding, these interactions are apparently removed, thereby allowing holo-IscR to bind both type 1 and type 2 motifs. These data suggest a unique mechanism of ligand-mediated DNA site recognition, whereby metallocluster ligation relocates a protein-specificity determinant to expand DNA target-site selection, allowing a broader transcriptomic response by holo-IscR.

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Coordinated conformational and compositional dynamics drive ribosome translocation.

Publication Date: 2013 Jun PMID: 23624862
Authors: Chen, J. - Petrov, A. - Tsai, A. - O'Leary, S. E. - Puglisi, J. D.
Journal: Nat Struct Mol Biol

During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G-GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G-GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.

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Structural basis for substrate transport in the GLUT-homology family of monosaccharide transporters.

Publication Date: 2013 Jun PMID: 23624861
Authors: Quistgaard, E. M. - Low, C. - Moberg, P. - Tresaugues, L. - Nordlund, P.
Journal: Nat Struct Mol Biol

Here we present two structures of the major facilitator (MFS) xylose transporter XylE from Escherichia coli in inward open and partially occluded inward open conformations. These structures provide key information about the transport cycle of XylE and the closely related human GLUT transporters. This is, to our knowledge, the first MFS transporter structure determined in more than one conformational state, which may establish XylE as an important MFS model protein.

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Substrate-specific structural rearrangements of human Dicer.

Publication Date: 2013 Jun PMID: 23624860
Authors: Taylor, D. W. - Ma, E. - Shigematsu, H. - Cianfrocco, M. A. - Noland, C. L. - Nagayama, K. - Nogales, E. - Doudna, J. A. - Wang, H. W.
Journal: Nat Struct Mol Biol

Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer-RNA complexes to gain insight into the structural basis for human Dicer's substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer.

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The microtubule-associated tau protein has intrinsic acetyltransferase activity.

Publication Date: 2013 Jun PMID: 23624859
Authors: Cohen, T. J. - Friedmann, D. - Hwang, A. W. - Marmorstein, R. - Lee, V. M.
Journal: Nat Struct Mol Biol

Tau proteins are the building blocks of neurofibrillary tangles (NFTs) found in a range of neurodegenerative tauopathies, including Alzheimer's disease. Recently, we demonstrated that tau is extensively post-translationally modified by lysine acetylation, which impairs normal tau function and promotes pathological aggregation. Identifying the enzymes that mediate tau acetylation could provide targets for future therapies aimed at reducing the burden of acetylated tau. Here, we report that mammalian tau proteins possess intrinsic enzymatic activity capable of catalyzing self-acetylation. Functional mapping of tau acetyltransferase activity followed by biochemical analysis revealed that tau uses catalytic cysteine residues in the microtubule-binding domain to facilitate tau lysine acetylation, thus suggesting a mechanism similar to that employed by MYST-family acetyltransferases. The identification of tau as an acetyltransferase provides a framework to further understand tau pathogenesis and highlights tau enzymatic activity as a potential therapeutic target.

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