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1.  The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation 
One X chromosome, selected at random, is silenced in each female mammalian cell. Xist encodes a noncoding RNA that influences the probability that the cis-linked X chromosome will be silenced. We found that the A-repeat, a highly conserved element within Xist, is required for the accumulation of spliced Xist RNA. In addition, the A-repeat is necessary for X-inactivation to occur randomly. In combination, our data suggest that normal Xist RNA processing is important in the regulation of random X-inactivation. We propose that modulation of Xist RNA processing may be part of the stochastic process that determines which X chromosome will be inactivated.
PMCID: PMC4336797  PMID: 20657585
2.  The ABCs of the ribosome 
PMCID: PMC4324477  PMID: 24500425
3.  Structure of human mitochondrial RNA polymerase elongation complex 
Nature structural & molecular biology  2013;20(11):1298-1303.
The crystal structure of the human mitochondrial RNA polymerase (mtRNAP) transcription elongation complex was determined at 2.65 Å resolution. The structure reveals a 9–base pair hybrid formed between the DNA template and the RNA transcript and one turn of DNA both upstream and downstream of the hybrid. Comparisons with the distantly related RNAP from bacteriophage T7 indicates conserved mechanisms for substrate binding and nucleotide incorporation, but also strong mechanistic differences. Whereas T7 RNAP refolds during the transition from initiation to elongation, mtRNAP adopts an intermediary conformation that is capable of elongation without refolding. The intercalating hairpin that melts DNA during T7 RNAP initiation separates RNA from DNA during mtRNAP elongation. Newly synthesized RNA exits towards the PPR domain, a unique feature of mtRNAP with conserved RNA recognition motifs.
PMCID: PMC4321815  PMID: 24096365
4.  RPRD1A and RPRD1B Are Human RNA Polymerase II C-Terminal Domain Scaffolds for Ser5 Dephosphorylation 
The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.
PMCID: PMC4124035  PMID: 24997600
5.  Identification of genes in trinucleotide repeat RNA toxicity pathways in C. elegans 
Myotonic dystrophy disorders are caused by expanded CUG repeats in non-coding regions. To reveal mechanisms of CUG repeat pathogenesis we used C. elegans expressing CUG repeats to identify gene inactivations that modulate CUG repeat toxicity. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and modulate formation of nuclear RNA foci by CUG repeats. These genes regulated CUG repeat-induced toxicity through distinct mechanisms including RNA export and RNA clearance, suggesting that CUG repeat toxicity is mediated by multiple pathways. A subset is shared with other degenerative disorders. The nonsense-mediated mRNA decay (NMD) pathway plays a conserved role regulating CUG repeat RNA transcript levels and toxicity, and NMD recognition of toxic RNAs depends on 3′UTR GC nucleotide content. Our studies suggest a broader surveillance role for NMD where variations in this pathway influence multiple degenerative diseases.
PMCID: PMC4125460  PMID: 25038802
CUG repeats; myotonic dystrophy; DM1; RNA toxicity; repeat disorders; nonsense-mediated decay; Caenorhabditis elegans
6.  A protein-RNA specificity code enables targeted activation of an endogenous human transcript 
Programmable protein scaffolds that target DNA are invaluable tools for genome engineering and designer control of transcription. RNA manipulation provides broad new opportunities for control, including changes in translation. PUF proteins are an attractive platform for that purpose, as they bind specific single-stranded RNA sequences using short repeated modules, each contributing three amino acids that contact an RNA base. Here, we identify the specificities of natural and designed combinations of those three amino acids, using a large randomized RNA library. The resulting specificity code reveals the RNA binding preferences of natural proteins and enables the design of new specificities. Using the code and a translational activation domain, we design a protein that targets endogenous cyclin B1 mRNA in human cells, increasing sensitivity to chemotherapeutic drugs. Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.
PMCID: PMC4125476  PMID: 24997599
7.  The ROQ domain of Roquin recognizes mRNA constitutive decay element and double-stranded RNA 
A conserved stem-loop motif of the constitutive decay element (CDE) in the 3′ UTR of mRNAs is recognized by the ROQ domain of Roquin, which mediates their degradation. Here we report two crystal structures of the Homo sapiens ROQ domain in complex with CDE RNA. The ROQ domain has an elongated shape, with three sub-domains. The 19-nt Hmgxb3 CDE is bound as a stem-loop to domain III. The 23-nt TNF RNA is bound as a duplex, to a separate site at the interface between domains I and II. Mutagenesis studies confirm that the ROQ domain has two separate RNA binding sites, one for stem-loop RNA (A site) and the other for dsRNA (B site). Mutation in either site perturbs the Roquin-mediated degradation of HMGXB3 and IL-6 mRNAs in human cells, demonstrating the importance of both sites for mRNA decay.
PMCID: PMC4125485  PMID: 25026078
8.  Molecular Basis for Unidirectional Scaffold Switching of Human Plk4 in Centriole Biogenesis 
Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. Here we showed that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses revealed that Cep192 - and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152-peptide bound to the CPB markedly better than the Cep192-peptide and effectively snatched the CPB away from a preformed CPB–Cep192-peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.
PMCID: PMC4125498  PMID: 24997597
9.  RAD51 and MRE11 dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks 
In higher eukaryotes the dynamics of replisome components during fork collapse and restart are poorly understood. Here, we reconstituted replication fork collapse and restart by inducing single-strand DNA (ssDNA) lesions that create a double-strand break (DSB) in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45–MCM–GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and Pol epsilon onto DNA in a RAD51- and MRE11- dependent manner, but independently of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results reveal that in higher eukaryotes replisomes are partially dismantled following fork collapse and fully re-established by a recombination-mediated process.
PMCID: PMC4306020  PMID: 22139015
10.  Rad51 protects nascent DNA from Mre11 dependent degradation and promotes continuous DNA synthesis 
Nature structural & molecular biology  2010;17(11):1305-1311.
The role of Rad51 in an unperturbed cell cycle has been difficult to dissect from its DNA repair function. Here, using electron microscopy (EM) to visualize replication intermediates (RIs) assembled in Xenopus laevis egg extract we show that Rad51 is required to prevent the accumulation of ssDNA gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading and lagging strand DNA synthesis. Instead, ssDNA gaps behind forks, which are exacerbated on damaged templates, result from Mre11 dependent degradation of newly synthesized DNA strands as they can be suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct and unanticipated roles for Rad51 at replication forks demonstrating that Rad51 protects newly synthesised DNA from Mre11 dependent degradation and promotes continuous DNA synthesis.
PMCID: PMC4306207  PMID: 20935632
11.  Transcriptionally active chromatin recruits homologous recombination at DNA double strand breaks 
While both Homologous recombination (HR) and Non Homologous End Joining (NHEJ) can repair DNA double Strand Breaks (DSB), the mechanisms by which one or other of these pathways is chosen remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq), to analyse 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 targeted to HR repair via the transcription-elongation associated histone mark, histone H3 lysine 36 trimethylation (H3K36me3). In agreement, depletion of SETD2, the main H3K36 tri-methyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role of the chromatin context, in which a break occurs, in DSB repair.
PMCID: PMC4300393  PMID: 24658350
DSB repair; NHEJ; HR; chromatin; transcription; ChIP-seq; H3K36me3
12.  Substrate binding on the APC/C occurs between the co-activator CDH1 and the processivity factor DOC1 
The anaphase–promoting complex/cyclosome (APC/C) is a 22 S ubiquitin ligase complex that initiates chromosome segregation and mitotic exit. We have used biochemical and electron microscopic analyses of Saccharomyces cerevisiae and human APC/C to address how the APC/C subunit Doc1 contributes to recruitment and processive ubiquitylation of APC/C substrates, and to understand how APC/C monomers interact to form a 36 S dimeric form. We show that Doc1 interacts with Cdc27, Cdc16 and Apc1, and is located in vicinity of the cullin–RING module Apc2–Apc11 in the inner cavity of the APC/C. Substrate proteins also bind in the inner cavity, in close proximity to DOC1 and the co–activator CDH1, and induce conformational changes in APC2–APC11. Our results suggest that substrates are recruited to the APC/C by binding to a bipartite substrate receptor composed of a co–activator protein and Doc1.
PMCID: PMC4300845  PMID: 21186364
13.  Translation drives mRNA quality control 
There are three predominant forms of co-translational mRNA surveillance: nonsense-mediated decay (NMD), no-go decay (NGD) and non-stop decay (NSD). While discussion of these pathways often focuses on mRNA fate, there is growing consensus that there are other important outcomes of these processes that must be simultaneously considered. Here, we seek to highlight similarities between NMD, NGD and NSD and their likely origins on the ribosome during translation.
PMCID: PMC4299859  PMID: 22664987
14.  [No title available] 
PMCID: PMC4292918  PMID: 24509834
15.  Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix 
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly-abundant nuclear long noncoding RNA that promotes malignancy. A 3′-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. 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. Interruption of the triple helix by the C-G doublet induces a “helical reset” that explains why triple-helical stacks longer than six do not occur in nature.
PMCID: PMC4096706  PMID: 24952594
16.  The translational landscape of fission yeast meiosis and sporulation 
Sexual development in the fission yeast Schizosaccharomyces pombe culminates in meiosis and sporulation. We used ribosome profiling to investigate the translational landscape of this process. We show that the translation efficiency of hundreds of genes is regulated in complex patterns, often correlating with changes in RNA levels. Ribosome-protected fragments show a three-nucleotide periodicity that identifies translated sequences and their reading frame. Using this property, we identified 46 novel translated genes and found that 24% of non-coding RNAs are actively translated. We also detected 19 nested antisense genes, in which both DNA strands encode translated mRNAs. Finally, we identified 1,735 translated upstream ORFs in leader sequences. In contrast with Saccharomyces cerevisiae, sexual development in S. pombe is not accompanied by large increases in upstream ORF use, suggesting that this is an organism-specific adaptation and not a general feature of developmental processes.
PMCID: PMC4082408  PMID: 24929437
17.  Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion 
Nucleosomes are the fundamental unit of chromatin, but the analysis of transcription-independent nucleosome functions has been thwarted by the confounding gene expression changes resultant of histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation, and analyze the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that while nucleosome-free DNA can recruit nuclear envelope membranes, nucleosomes are required for spindle assembly, lamina and nuclear pore complex (NPC) formation. In addition to RCC1, we reveal that ELYS, the initiator of NPC formation, fails to associate with naked DNA, but directly binds histones H2A–H2B and nucleosomes. Tethering ELYS and RCC1 to DNA bypassed the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.
PMCID: PMC4082469  PMID: 24952593
18.  Evidence for a group II intron-like catalytic triplex in the spliceosome 
To catalyze pre-mRNA splicing, U6 snRNA positions two metals that interact directly with the scissile phosphates. The U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. In domain V, the ligands are organized by base-triple interactions, which also juxtapose the 3′ splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, crosslinking, and biochemistry in yeast that analogous triples form in U6 and promote catalytic metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5′ splice site, the triples also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.
PMCID: PMC4257784  PMID: 24747940
19.  Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity 
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-Å 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.
PMCID: PMC4075942  PMID: 24793649
Dicer is a central enzymatic player in RNA interference (RNAi) pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well-documented in mammals, its nuclear function remains obscure. Here we show that Dicer is present in both the nucleus and cytoplasm, but that its nuclear levels are tightly regulated. In its nuclear manifestation, Dicer interacts with RNA polymerase II (Pol II) at actively-transcribed gene loci. Loss of Dicer causes the appearance of endogenous dsRNA, leading to induction of the interferon response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping non-coding RNA transcription units. Failure to do so has catastrophic effects on cell function.
PMCID: PMC4129937  PMID: 24814348
21.  The DNA-Binding Domain Mediates both Nuclear and Cytosolic Functions of p53 
Under conditions of genotoxic stress, human p53 activates the apoptotic effectors BAX or BAK, resulting in mitochondrial outer membrane permeabilization and apoptosis. Anti-apoptotic BCL-2 family member BCL-xL opposes this activity by sequestering cytosolic p53 via association with its DNA-binding domain, an interaction that is enhanced by p53 tetramerization. Here we characterized the BCL-xL – p53 complex using NMR spectroscopy and modulated it through mutagenesis to determine the relative contributions of BCL-xL’s interactions with p53, or with other BCL-2 family proteins, to BCL-xL-dependent inhibition of UV irradiation-induced apoptosis. Under our experimental conditions, one third of the anti-apoptotic activity of BCL-xL was mediated by p53 sequestration and the remaining two thirds through sequestration of pro-apoptotic BCL-2 family members. Our studies define the contributions of cytosolic p53 to UV irradiation-induced apoptosis and provide opportunities to explore its contributions to other, p53-dependent apoptotic signaling pathways.
PMCID: PMC4134560  PMID: 24814347
22.  Core structure of the U6 snRNP at 1.7 Å resolution 
The spliceosome is a dynamic assembly of five small nuclear ribonucleoproteins (snRNPs) that removes introns from eukaryotic pre-mRNA. U6 is the most conserved of the spliceosomal snRNAs and participates directly in catalysis. Here, we report the crystal structure of the Saccharomyces cerevisiae U6 snRNP core, containing most of U6 snRNA and all four RRM domains of the Prp24 protein. It reveals a unique interlocked RNP architecture that sequesters the 5′ splice site-binding bases of U6 snRNA. RRMs 1, 2 and 4 of Prp24 form an electropositive groove that binds double-stranded RNA and may nucleate annealing of U4 and U6 snRNAs. Substitutions in Prp24 that suppress a mutation in U6 localize to direct RNA-protein contacts. Our results provide the most complete view to date of a multi-RRM protein bound to RNA, and reveal striking co-evolution of protein and RNA structure.
PMCID: PMC4141773  PMID: 24837192
23.  PTEN is a protein tyrosine phosphatase for IRS1 
The biological function of the PTEN tumor suppressor is mainly attributed to its lipid phosphatase activity. This study demonstrates that mammalian PTEN is a protein tyrosine phosphatase that selectively dephosphorylates insulin receptor substrate-1 (IRS1), a mediator of insulin and IGF signals. IGF signaling was defective in cells lacking NEDD4, a PTEN ubiquitin ligase, whereas AKT activation triggered by EGF or serum was unimpaired. Defective IGF signaling caused by NEDD4 deletion, including phosphorylation of IRS1 and AKT, was rescued by PTEN ablation. We demonstrate the nature of PTEN as an IRS1 phosphatase by direct biochemical analysis and cellular reconstitution, showing that NEDD4 supports insulin-mediated glucose metabolism and is required for the proliferation of IGF1 receptor–dependent but not EGF receptor–dependent tumor cells. Thus, PTEN is a protein phosphatase for IRS1, and its antagonism by NEDD4 promotes signaling by IGF and insulin.
PMCID: PMC4167033  PMID: 24814346
24.  Cks Confers Specificity to Phosphorylation-Dependent Cdk Signaling Pathways 
Nature structural & molecular biology  2013;20(12):1407-1414.
Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (Cdk) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical, as Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding yeast Cks associates with select phosphorylated sequences in cell cycle regulatory proteins. We characterize the molecular interactions responsible for this specificity and demonstrate that Cks enhances Cdk activity in response to specific priming phosphosites. Identification of the binding consensus sequence allows us to identify putative Cks-directed Cdk substrates and binding partners. We characterize novel Cks binding sites in the mitotic regulator Wee1 and discover a novel role for Cks in regulating Cdk activity at mitotic entry. Together our results portray Cks as a multifunctional phosphoadaptor that serves as a specificity factor for Cdk activity.
PMCID: PMC4242096  PMID: 24186063
25.  Temporal orchestration of repressive chromatin modifiers by mammalian circadian clock PERIOD complexes 
The mammalian circadian clock is built on a molecular feedback loop in which the PERIOD (PER) proteins, acting in a large, poorly-understood complex, repress CLOCK-BMAL1, the transcription factor driving their expression. We found that mouse PER complexes include the histone methyltransferase HP1γ-Suv39H. PER proteins recruit HP1γ-Suv39H to the Per1 and Per2 promoters, and HP1γ-Suv39H proved important for circadian di- and tri-methylation of histone-3 lysine-9 (H3K9) at the Per1 promoter, feedback transcriptional repression, and circadian clock function. HP1γ-Suv39H was recruited to the Per1 and Per2 promoters ~4 hours after HDAC1, a PER-associated protein we previously implicated in clock function and H3K9 deacetylation at the Per1 promoter. PER complexes containing HDAC1 or HP1γ-Suv39H appeared to be physically separable. Circadian clock negative feedback by the PER complex thus involves dynamic, ordered recruitment of repressive chromatin modifiers to DNA-bound CLOCK-BMAL1.
PMCID: PMC4227600  PMID: 24413057

Results 1-25 (830)