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Showing papers on "Transcription (biology) published in 2014"


Journal ArticleDOI
27 Mar 2014-Cell
TL;DR: The pathway of ncRNA research is described, where every established "rule" seems destined to be overturned.

1,875 citations


Journal ArticleDOI
TL;DR: In this paper, two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyl transferase like 14 (METTL14), were reported.
Abstract: The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

1,516 citations


Journal ArticleDOI
21 Mar 2014-Science
TL;DR: FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.
Abstract: Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

887 citations


Journal ArticleDOI
06 Nov 2014-Nature
TL;DR: Gilbert et al. as discussed by the authors used pseudouridylation detection on a genome-wide scale, leading to the identification of pseudouridine in messenger RNAs as well as almost 100 new sites in non-coding RNAs.
Abstract: The modification of uridine to pseudouridine is widespread in transfer and ribosomal RNAs but not observed so far in a coding RNA; here a new technique is used to detect this modification on a genome-wide scale, leading to the identification of pseudouridylation in messenger RNAs as well as almost 100 new sites in non-coding RNAs. The modification of uridine to pseudouridine is widespread in transfer and ribosomal RNAs. Until now it had not been identified in a coding RNA. Wendy Gilbert and colleagues have employed a next-generation sequencing technique called Pseudo-seq to measure the uridine-to-pseudouridine modification on a genome-wide scale. They not only identify almost 100 new sites in non-coding RNAs, but also discover pseudouridylation in mRNAs. These modifications are mostly due to the activity of the Pus pseudouridine synthase family members, and they can be responsive to environmental conditions, suggesting that inducible modification of RNA might be another way to regulate transcription. Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure2,3,4,5,6,7,8. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function—it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11,12,13.

733 citations


Journal ArticleDOI
02 Oct 2014-Nature
TL;DR: The role of long noncoding RNA (lncRNA) in adult heart is unknown; also unclear is how lncRNA modulates nucleosome remodelling as discussed by the authors. But they identify a cluster of LncRNA transcripts from myosin heavy chain 7 (Myh7) loci and demonstrate a new lnc RNA-chromatin mechanism for heart failure.
Abstract: The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA-chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized--but not naked--DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA-chromatin interaction.

610 citations


Journal ArticleDOI
05 Sep 2014-Science
TL;DR: It is shown that the peptides encoded by the expanded repeats in the C9orf72 gene interfere with the way cells make RNA and kill cells, providing evidence that dipeptide repeat proteins can cause toxicity directly.
Abstract: Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

534 citations


Journal ArticleDOI
04 Sep 2014-Nature
TL;DR: An eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds is reported.
Abstract: The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5′ untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5′ UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase. The translation of many messenger RNAs that encode important oncogenes and transcription factors depends on the eIF4A RNA helicase to resolve G-quadruplex structures, implying eIF4A inhibition as an effective cancer therapy. The expression of some oncoproteins is regulated at the translational level. Hans-Guido Wendel and colleagues show that a subset of oncoprotein- and transcription factor-encoding mRNAs that are dependent on the translation initiation factor eIF4A contain a G-quadruplex-forming structure in their 5′ untranslated regions. These findings explain why silvestrol, a plant-derived anticancer agent that targets eIF4A-dependent translation, is not generally toxic but can be well tolerated except in cancer cells which are dependent on the activities of these proteins. In a separate study in this issue, Stephan Vagner and colleagues show that inhibition of eIF4F cooperates with BRAF inhibitors in reducing the growth of melanomas linked to BRAF mutations.

493 citations


Journal ArticleDOI
TL;DR: This study reveals that the SARS-coronavirus RNA polymerase (nsp12) needs to associate with nsp7 and nsp8 to activate its capability to replicate long RNA and defines the core of an RNA-synthesizing machinery that is unique in the RNA virus world and includes several key targets for antiviral drug development.
Abstract: In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

486 citations


Journal ArticleDOI
TL;DR: In this article, inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions and restoring the expression of editing-active cytoplasmic ADARs.

467 citations


Journal ArticleDOI
02 Jul 2014-Nature
TL;DR: The results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer–promoter loops through release of paused polymerase.
Abstract: Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.

461 citations


01 Jun 2014
TL;DR: In this article, a covalent cyclin-dependent kinase inhibitor, THZ1, was proposed to target a remote cysteine residue located outside of the canonical kinase domain.
Abstract: Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.

Journal ArticleDOI
TL;DR: The H19 long noncoding RNA has a critical trans-regulatory function in skeletal muscle differentiation and regeneration that is mediated by the microRNAs encoded within H19.
Abstract: Regulated expression of the H19 long noncoding RNA gene has been well characterized as a paradigm for genomic imprinting, but the H19 RNA's biological function remains largely unclear. H19 is abundantly expressed maternally in embryonic tissues but is strongly repressed after birth, and significant transcription persists only in skeletal muscle. Thus, we examined the role of the H19 RNA in skeletal muscle differentiation and regeneration. Knockdown of H19 RNA in myoblast cells and H19 knockout mouse satellite cells decreases differentiation. H19 exon1 encodes two conserved microRNAs, miR-675-3p and miR-675-5p, both of which are induced during skeletal muscle differentiation. The inhibition of myogenesis by H19 depletion during myoblast differentiation is rescued by exogenous expression of miR-675-3p and miR-675-5p. H19-deficient mice display abnormal skeletal muscle regeneration after injury, which is rectified by reintroduction of miR-675-3p and miR-675-5p. miR-675-3p and miR-675-5p function by directly targeting and down-regulating the anti-differentiation Smad transcription factors critical for the bone morphogenetic protein (BMP) pathway and the DNA replication initiation factor Cdc6. Therefore, the H19 long noncoding RNA has a critical trans-regulatory function in skeletal muscle differentiation and regeneration that is mediated by the microRNAs encoded within H19.

Journal ArticleDOI
24 Jul 2014-Nature
TL;DR: The results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.
Abstract: Global transcriptional and epigenomic analyses in diverse cell types reveal that the primary action of Myc is to up- and downregulate transcription of distinct groups of genes, rather than to amplify transcription of all active genes; general RNA amplification, when observed, is better explained as an indirect consequence of Myc’s action on cellular physiology. The mammalian Myc oncoprotein is a transcription factor that binds to thousands of promoters. Two current models for Myc function propose that it is either a gene-specific regulator of transcription, or a global amplifier of all active genes. Two groups reporting in this issue of Nature present evidence in support of the idea that Myc regulates specific genes. Arianna Sabo et al. analyse Myc genomic distribution and RNA expression profiles during B-cell lymphomagenesis in mice and Susanne Walz et al. compare normal cells and Myc-transformed tumour cells. Although both groups find that Myc overexpression can result in a general increase in gene expression, the effect is an indirect one. Modulated by various other transcription factors, Myc seems to act primarily by regulating specific groups of genes. The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci1. Recent work suggested that rather than up- and downregulating selected groups of genes1,2,3, Myc targets all active promoters and enhancers in the genome (a phenomenon termed ‘invasion’) and acts as a general amplifier of transcription4,5. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations6, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed ‘amplification’)4,5 when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response)7,8 or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome4,5,9,10,11, Myc does not directly act as a global transcriptional amplifier4,5. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

Journal ArticleDOI
TL;DR: It is shown that PRC2 activity is not only associated with H3K27me3 but also regulates all forms of H3k27 methylation in a spatially defined manner, contributing to different genomic functions in mouse embryonic stem cells.

Journal ArticleDOI
18 Dec 2014-Nature
TL;DR: The crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter is presented, laying the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase and opens new opportunities for anti-influenza drug design.
Abstract: The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3′ and 5′ extremities of the viral RNA. Here we present the crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. PB1 contains a canonical RNA polymerase fold that is stabilized by large interfaces with PA and PB2. The PA endonuclease and the PB2 cap-binding domain, involved in transcription by cap-snatching, form protrusions facing each other across a solvent channel. The 5′ extremity of the promoter folds into a compact hook that is bound in a pocket formed by PB1 and PA close to the polymerase active site. This structure lays the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase, and opens new opportunities for anti-influenza drug design. The crystal structure of the bat-specific influenza A polymerase in complex with the viral RNA promoter is presented, revealing how binding of the 5′ end of the viral RNA is required to activate or enhance the polymerase allosterically. Stephen Cusack and colleagues have solved the crystal structure of the complete influenza polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. In the first of two papers they present the structure of the polymerase from a bat-specific influenza A virus, which is evolutionarily close to human/avian influenza A strains. The second paper presents the structure of the polymerase from a human isolate of influenza B. Together, the structures provide a wealth of information about how the influenza polymerase functions and how the different subunits interact with each other.

Journal ArticleDOI
14 Aug 2014-Cell
TL;DR: It is shown that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation, and forced chromatin looping can override a stringent gene expression program.

Journal ArticleDOI
TL;DR: Paraspeckles are subnuclear structures formed around NEAT1 lncRNA that affect the transcription of several genes, arguing for a novel role for lnc RNA in gene regulation.
Abstract: Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENe/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno-electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1(-/-) fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.

Journal ArticleDOI
17 Jul 2014-Cell
TL;DR: It is shown that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation, and proves that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to aDNA segment, changing the supercoiled level of the segment.


Journal ArticleDOI
TL;DR: It is found that the entry site of the first (+1) nucleosome is a barrier to RNAPII for essentially all genes, including those undergoing regulated pausing farther upstream, and that depletion of H2A.Z from a nucleosomes position results in a higher barrier toRNAPII.

Journal ArticleDOI
15 Aug 2014-Science
TL;DR: An architecture for designing artificial RNA structures that fold from a single strand is introduced, in which arrays of antiparallel RNA helices are precisely organized by RNA tertiary motifs and a new type of crossover pattern.
Abstract: Artificial DNA and RNA structures have been used as scaffolds for a variety of nanoscale devices. In comparison to DNA structures, RNA structures have been limited in size, but they also have advantages: RNA can fold during transcription and thus can be genetically encoded and expressed in cells. We introduce an architecture for designing artificial RNA structures that fold from a single strand, in which arrays of antiparallel RNA helices are precisely organized by RNA tertiary motifs and a new type of crossover pattern. We constructed RNA tiles that assemble into hexagonal lattices and demonstrated that lattices can be made by annealing and/or cotranscriptional folding. Tiles can be scaled up to 660 nucleotides in length, reaching a size comparable to that of large natural ribozymes.

Journal ArticleDOI
TL;DR: It is shown that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development.

Journal ArticleDOI
TL;DR: It is shown that specific activation of either MRTFs or TCFs can reset the circadian clock, underpinning the role of SRF in cytoskeletal dynamics and mechanosensing.
Abstract: The transcription factor SRF (serum response factor) recruits two families of coactivators, the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors), to couple gene transcription to growth factor signaling. Here we investigated the role of the SRF network in the immediate transcriptional response of fibroblasts to serum stimulation. SRF recruited its cofactors in a gene-specific manner, and virtually all MRTF binding was directed by SRF. Much of SRF DNA binding was serum-inducible, reflecting a requirement for MRTF–SRF complex formation in nucleosome displacement. We identified 960 serum-responsive SRF target genes, which were mostly MRTF-controlled, as assessed by MRTF chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) and/or sensitivity to MRTF-linked signals. MRTF activation facilitates RNA polymerase II (Pol II) recruitment or promoter escape according to gene context. MRTF targets encode regulators of the cytoskeleton, transcription, and cell growth, underpinning the role of SRF in cytoskeletal dynamics and mechanosensing. Finally, we show that specific activation of either MRTFs or TCFs can reset the circadian clock.

Journal ArticleDOI
18 Dec 2014-Nature
TL;DR: It is predicted that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes.
Abstract: The formation of R-loops is a natural consequence of the transcription process, caused by invasion of the DNA duplex by nascent transcripts. These structures have been considered rare transcriptional by-products with potentially harmful effects on genome integrity owing to the fragility of the displaced DNA coding strand. However, R-loops may also possess beneficial effects, as their widespread formation has been detected over CpG island promoters in human genes. Furthermore, we have previously shown that R-loops are particularly enriched over G-rich terminator elements. These facilitate RNA polymerase II (Pol II) pausing before efficient termination. Here we reveal an unanticipated link between R-loops and RNA-interference-dependent H3K9me2 formation over pause-site termination regions in mammalian protein-coding genes. We show that R-loops induce antisense transcription over these pause elements, which in turn leads to the generation of double-stranded RNA and the recruitment of DICER, AGO1, AGO2 and the G9a histone lysine methyltransferase. Consequently, an H3K9me2 repressive mark is formed and heterochromatin protein 1γ (HP1γ) is recruited, which reinforces Pol II pausing before efficient transcriptional termination. We predict that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes.

Journal ArticleDOI
10 Apr 2014-Nature
TL;DR: This study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression.
Abstract: Candidate tumour suppressor ZMYND11 specifically recognizes histone K36 trimethylation on the histone variant H33 and helps regulate transcription elongation This study identifies the PHD–bromo–PWWP cassette of the candidate tumour suppressor ZMYND11 as a specific 'reader' of trimethylated K36 on the histone variant H33 ZMYND11 seems to accumulate on gene bodies during transcription and functions in modulating transcription elongation Structural and genomics experiments reveal the chromatin-binding properties of ZMYND11, and it is shown to be important for repressing transcription of genes linked to tumour cell growth Recognition of modified histones by ‘reader’ proteins plays a critical role in the regulation of chromatin1 H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions after RNA polymerase II elongation In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state, thus suppressing cryptic transcription2 However, much less is known about the role of H3K36me3 in transcription regulation in mammals This is further complicated by the transcription-coupled incorporation of the histone variant H33 in gene bodies3 Here we show that the candidate tumour suppressor ZMYND11 specifically recognizes H3K36me3 on H33 (H33K36me3) and regulates RNA polymerase II elongation Structural studies show that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H33-dependent recognition is mediated by the encapsulation of the H33-specific ‘Ser 31’ residue in a composite pocket formed by the tandem bromo–PWWP domains of ZMYND11 Chromatin immunoprecipitation followed by sequencing shows a genome-wide co-localization of ZMYND11 with H3K36me3 and H33 in gene bodies, and its occupancy requires the pre-deposition of H33K36me3 Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription co-repressor by modulating RNA polymerase II at the elongation stage ZMYND11 is critical for the repression of a transcriptional program that is essential for tumour cell growth; low expression levels of ZMYND11 in breast cancer patients correlate with worse prognosis Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumour formation in mice Together, this study identifies ZMYND11 as an H33-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression

Journal ArticleDOI
TL;DR: Inforna was applied to all human microRNA precursors and identified bioactive small molecules that inhibit biogenesis by binding to nuclease processing sites (41% hit rate) and shows that 1 only significantly effects microRNA-96 biogenesis and is more selective than an oligonucleotide.
Abstract: Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

Journal ArticleDOI
24 Jan 2014-Science
TL;DR: A transgenic mouse with fluorescently labeled endogenous β-actin mRNA permits single-molecule analysis in live cells and provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.
Abstract: The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

Journal ArticleDOI
TL;DR: The Y‐box binding protein 1 (YB‐1, YBX1) is a member of the family of DNA‐ and RNA‐binding proteins with an evolutionarily ancient and conserved cold shock domain that falls into a group of intrinsically disordered proteins that introduce a novel principle stating that a disordered structure suggests many functions.
Abstract: The Y-box binding protein 1 (YB-1, YBX1) is a member of the family of DNA- and RNA-binding proteins with an evolutionarily ancient and conserved cold shock domain. It falls into a group of intrinsically disordered proteins that do not follow the classical rule 'one protein-one function' but introduce a novel principle stating that a disordered structure suggests many functions. YB-1 participates in a wide variety of DNA/RNA-dependent events, including DNA reparation, pre-mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. At the cell level, the multiple activities of YB-1 are manifested as its involvement in cell proliferation and differentiation, stress response, and malignant cell transformation. WIREs RNA 2014, 5:95-110. doi: 10.1002/wrna.1200 CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

Journal ArticleDOI
TL;DR: E ectopically expressed NRAV in human cells or transgenic mice and found that it significantly promotes influenza A virus replication and virulence and silencing NRAV suppressed IAV replication and virus production, suggesting that reduction of NRAV is part of the host antiviral innate immune response to virus infection.

Journal ArticleDOI
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.