A synthetic DNA-binding inhibitor of SOX2 guides human induced pluripotent stem cells to differentiate into mesoderm Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-31 Junichi Taniguchi, Ganesh N. Pandian, Takuya Hidaka, Kaori Hashiya, Toshikazu Bando, Kyeong Kyu Kim, Hiroshi Sugiyama
Targeted differentiation of human induced pluripotent stem cells (hiPSCs) using only chemicals would have value-added clinical potential in the regeneration of complex cell types including cardiomyocytes. Despite the availability of several chemical inhibitors targeting proteins involved in signaling pathways, no bioactive synthetic DNA-binding inhibitors, targeting key cell fate-controlling genes such as SOX2, are yet available. Here, we demonstrate a novel DNA-based chemical approach to guide the differentiation of hiPSCs using pyrrole–imidazole polyamides (PIPs), which are sequence-selective DNA-binding synthetic molecules. Harnessing knowledge about key transcriptional changes during the induction of cardiomyocyte, we developed a DNA-binding inhibitor termed PIP-S2, targeting the 5′-CTTTGTT-3′ and demonstrated that inhibition of SOX2–DNA interaction by PIP-S2 triggers the mesoderm induction in hiPSCs. Genome-wide gene expression analyses revealed that PIP-S2 induced mesoderm by targeted alterations in SOX2-associated gene regulatory networks. Also, employment of PIP-S2 along with a Wnt/β-catenin inhibitor successfully generated spontaneously contracting cardiomyocytes, validating our concept that DNA-binding inhibitors could drive the directed differentiation of hiPSCs. Because PIPs can be fine-tuned to target specific DNA sequences, our DNA-based approach could be expanded to target and regulate key transcription factors specifically associated with desired cell types.
Hydroxyl-radical footprinting combined with molecular modeling identifies unique features of DNA conformation and nucleosome positioning Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 Alexey K. Shaytan, Hua Xiao, Grigoriy A. Armeev, Carl Wu, David Landsman, Anna R. Panchenko
Nucleosomes are the most abundant protein–DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein–DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.
Automatic identification of informative regions with epigenomic changes associated to hematopoiesis Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 Enrique Carrillo-de-Santa-Pau, David Juan, Vera Pancaldi, Felipe Were, Ignacio Martin-Subero, Daniel Rico, Alfonso Valencia
Hematopoiesis is one of the best characterized biological systems but the connection between chromatin changes and lineage differentiation is not yet well understood. We have developed a bioinformatic workflow to generate a chromatin space that allows to classify 42 human healthy blood epigenomes from the BLUEPRINT, NIH ROADMAP and ENCODE consortia by their cell type. This approach let us to distinguish different cells types based on their epigenomic profiles, thus recapitulating important aspects of human hematopoiesis. The analysis of the orthogonal dimension of the chromatin space identify 32,662 chromatin determinant regions (CDRs), genomic regions with different epigenetic characteristics between the cell types. Functional analysis revealed that these regions are linked with cell identities. The inclusion of leukemia epigenomes in the healthy hematological chromatin sample space gives us insights on the healthy cell types that are more epigenetically similar to the disease samples. Further analysis of tumoral epigenetic alterations in hematopoietic CDRs points to sets of genes that are tightly regulated in leukemic transformations and commonly mutated in other tumors. Our method provides an analytical approach to study the relationship between epigenomic changes and cell lineage differentiation. Method availability: https://github.com/david-juan/ChromDet.
CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-09 Zijun Zhang, Yi Xing
Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation–maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein–RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome.
Accumulation of histone variant H3.3 with age is associated with profound changes in the histone methylation landscape Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-04 Andrey Tvardovskiy, Veit Schwämmle, Stefan J. Kempf, Adelina Rogowska-Wrzesinska, Ole N. Jensen
Deposition of replication-independent histone variant H3.3 into chromatin is essential for many biological processes, including development and reproduction. Unlike replication-dependent H3.1/2 isoforms, H3.3 is expressed throughout the cell cycle and becomes enriched in postmitotic cells with age. However, lifelong dynamics of H3 variant replacement and the impact of this process on chromatin organization remain largely undefined. Using quantitative middle-down proteomics we demonstrate that H3.3 accumulates to near saturation levels in the chromatin of various mouse somatic tissues by late adulthood. Accumulation of H3.3 is associated with profound changes in global levels of both individual and combinatorial H3 methyl modifications. A subset of these modifications exhibit distinct relative abundances on H3 variants and remain stably enriched on H3.3 throughout the lifespan, suggesting a causal relationship between H3 variant replacement and age-dependent changes in H3 methylation. Furthermore, the H3.3 level is drastically reduced in human hepatocarcinoma cells as compared to nontumoral hepatocytes, suggesting the potential utility of the H3.3 relative abundance as a biomarker of abnormal cell proliferation activity. Overall, our study provides the first quantitative characterization of dynamic changes in H3 proteoforms throughout lifespan in mammals and suggests a role for H3 variant replacement in modulating H3 methylation landscape with age.
A comprehensive, cell specific microRNA catalogue of human peripheral blood Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-11 Simonas Juzenas, Geetha Venkatesh, Matthias Hübenthal, Marc P. Hoeppner, Zhipei Gracie Du, Maren Paulsen, Philip Rosenstiel, Philipp Senger, Martin Hofmann-Apitius, Andreas Keller, Limas Kupcinskas, Andre Franke, Georg Hemmrich-Stanisak
With this study, we provide a comprehensive reference dataset of detailed miRNA expression profiles from seven types of human peripheral blood cells (NK cells, B lymphocytes, cytotoxic T lymphocytes, T helper cells, monocytes, neutrophils and erythrocytes), serum, exosomes and whole blood. The peripheral blood cells from buffy coats were typed and sorted using FACS/MACS. The overall dataset was generated from 450 small RNA libraries using high-throughput sequencing. By employing a comprehensive bioinformatics and statistical analysis, we show that 3′ trimming modifications as well as composition of 3′ added non-templated nucleotides are distributed in a lineage-specific manner—the closer the hematopoietic progenitors are, the higher their similarities in sequence variation of the 3′ end. Furthermore, we define the blood cell-specific miRNA and isomiR expression patterns and identify novel cell type specific miRNA candidates. The study provides the most comprehensive contribution to date towards a complete miRNA catalogue of human peripheral blood, which can be used as a reference for future studies. The dataset has been deposited in GEO and also can be explored interactively following this link: http://188.8.131.52/ikmb-tools/bloodmiRs.
The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-15 Fernando Gómez-Herreros, Thanasis Margaritis, Olga Rodríguez-Galán, Vicent Pelechano, Victoria Begley, Gonzalo Millán-Zambrano, Macarena Morillo-Huesca, Mari Cruz Muñoz-Centeno, José E. Pérez-Ortín, Jesús de la Cruz, Frank C. P. Holstege, Sebastián Chávez
Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the up-regulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesis mutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.
Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-28 Paula A. Agudelo Garcia, Michael E. Hoover, Pei Zhang, Prabakaran Nagarajan, Michael A. Freitas, Mark R. Parthun
Histone acetyltransferase 1 (Hat1) catalyzes the acetylation of newly synthesized histone H4 at lysines 5 and 12 that accompanies replication-coupled chromatin assembly. The acetylation of newly synthesized H4 occurs in the cytoplasm and the function of this acetylation is typically ascribed to roles in either histone nuclear import or deposition. Using cell lines from Hat1+/+ and Hat1−/− mouse embryos, we demonstrate that Hat1 is not required for either histone nuclear import or deposition. We employed quantitative proteomics to characterize Hat1-dependent changes in the composition of nascent chromatin structure. Among the proteins depleted from nascent chromatin isolated from Hat1−/− cells are several bromodomain-containing proteins, including Brg1, Baz1A and Brd3. Analysis of the binding specificity of their bromodomains suggests that Hat1-dependent acetylation of H4 is directly involved in their recruitment. Hat1−/− nascent chromatin is enriched for topoisomerase 2α and 2β. The enrichment of topoisomerase 2 is functionally relevant as Hat1−/− cells are hyper-sensitive to topoisomerase 2 inhibition suggesting that Hat1 is required for proper chromatin topology. In addition, our results indicate that Hat1 is transiently recruited to sites of chromatin assembly, dissociating prior to the maturation of chromatin structure.
XIAP upregulates expression of HIF target genes by targeting HIF1α for Lys63-linked polyubiquitination Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-28 Catherine V. Park, Iglika G. Ivanova, Niall S. Kenneth
The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1α subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1α. Lys63-linked ubiquitination of HIF1α by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1α nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1α, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease.
Crosstalk between histone modifications indicates that inhibition of arginine methyltransferase CARM1 activity reverses HIV latency Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-20 Zheng Zhang, Bryan C. Nikolai, Leah A. Gates, Sung Yun Jung, Edward B. Siwak, Bin He, Andrew P. Rice, Bert W. O’Malley, Qin Feng
In eukaryotic cells, the gene expression status is strictly controlled by epigenetic modifications on chromatin. The repressive status of chromatin largely contributes to HIV latency. Studies have shown that modification of histone H3K27 acts as a key molecular switch for activation or suppression of many cellular genes. In this study, we found that K27-acetylated histone H3 specifically recruited Super Elongation Complex (SEC), the transcriptional elongation complex essential for HIV-1 long terminal repeat (LTR)-mediated and general cellular transcription. Interestingly, H3K27 acetylation further stimulates H3R26 methylation, which subsequently abrogates the recruitment of SEC, forming a negative feedback regulatory loop. Importantly, by inhibiting methyltransferase activity of CARM1, the enzyme responsible for H3R26 methylation, HIV-1 transcription is reactivated in several HIV latency cell models, including a primary resting CD4+ T cell model. When combined with other latency disrupting compounds such as JQ1 or vorinostat/SAHA, the CARM1 inhibitor achieved synergistic effects on HIV-1 activation. This study suggests that coordinated and dynamic modifications at histone H3K27 and H3R26 orchestrate HIV-1 LTR-mediated transcription, and potentially opens a new avenue to disrupt latent HIV-1 infection by targeting specific epigenetic enzymes.
Recruitment and delivery of the fission yeast Rst2 transcription factor via a local genome structure counteracts repression by Tup1-family corepressors Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-27 Ryuta Asada, Miki Umeda, Akira Adachi, Satoshi Senmatsu, Takuya Abe, Hiroshi Iwasaki, Kunihiro Ohta, Charles S. Hoffman, Kouji Hirota
Transcription factors (TFs) determine the transcription activity of target genes and play a central role in controlling the transcription in response to various environmental stresses. Three dimensional genome structures such as local loops play a fundamental role in the regulation of transcription, although the link between such structures and the regulation of TF binding to cis-regulatory elements remains to be elucidated. Here, we show that during transcriptional activation of the fission yeast fbp1 gene, binding of Rst2 (a critical C2H2 zinc-finger TF) is mediated by a local loop structure. During fbp1 activation, Rst2 is first recruited to upstream-activating sequence 1 (UAS1), then it subsequently binds to UAS2 (a critical cis-regulatory site located approximately 600 base pairs downstream of UAS1) through a loop structure that brings UAS1 and UAS2 into spatially close proximity. Tup11/12 (the Tup-family corepressors) suppress direct binding of Rst2 to UAS2, but this suppression is counteracted by the recruitment of Rst2 at UAS1 and following delivery to UAS2 through a loop structure. These data demonstrate a previously unappreciated mechanism for the recruitment and expansion of TF-DNA interactions within a promoter mediated by local three-dimensional genome structures and for timely TF-binding via counteractive regulation by the Tup-family corepressors.
Regulation of chromatin folding by conformational variations of nucleosome linker DNA Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-27 Jenna M. Buckwalter, Davood Norouzi, Anna Harutyunyan, Victor B. Zhurkin, Sergei A. Grigoryev
Linker DNA conformational variability has been proposed to direct nucleosome array folding into more or less compact chromatin fibers but direct experimental evidence for such models are lacking. Here, we tested this hypothesis by designing nucleosome arrays with A-tracts at specific locations in the nucleosome linkers to induce inward (AT-IN) and outward (AT-OUT) bending of the linker DNA. Using electron microscopy and analytical centrifugation techniques, we observed spontaneous folding of AT-IN nucleosome arrays into highly compact structures, comparable to those induced by linker histone H1. In contrast, AT-OUT nucleosome arrays formed less compact structures with decreased nucleosome interactions similar to wild-type nucleosome arrays. Adding linker histone H1 further increased compaction of the A-tract arrays while maintaining structural differences between them. Furthermore, restriction nuclease digestion revealed a strongly reduced accessibility of nucleosome linkers in the compact AT-IN arrays. Electron microscopy analysis and 3D computational Monte Carlo simulations are consistent with a profound zigzag linker DNA configuration and closer nucleosome proximity in the AT-IN arrays due to inward linker DNA bending. We propose that the evolutionary preferred positioning of A-tracts in DNA linkers may control chromatin higher-order folding and thus influence cellular processes such as gene expression, transcription and DNA repair.
Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-27 Yajuan Lu, Xiaoxin Dai, Mianqun Zhang, Yilong Miao, Changyin Zhou, Zhaokang Cui, Bo Xiong
Sister chromatid cohesion, mediated by cohesin complex and established by the acetyltransferases Esco1 and Esco2, is essential for faithful chromosome segregation. Mutations in Esco2 cause Roberts syndrome, a developmental disease characterized by severe prenatal retardation as well as limb and facial abnormalities. However, its exact roles during oocyte meiosis have not clearly defined. Here, we report that Esco2 localizes to the chromosomes during oocyte meiotic maturation. Depletion of Esco2 by morpholino microinjection leads to the precocious polar body extrusion, the escape of metaphase I arrest induced by nocodazole treatment and the loss of BubR1 from kinetochores, indicative of inactivated SAC. Furthermore, depletion of Esco2 causes a severely impaired spindle assembly and chromosome alignment, accompanied by the remarkably elevated incidence of defective kinetochore-microtubule attachments which consequently lead to the generation of aneuploid eggs. Notably, we find that the involvement of Esco2 in SAC and kinetochore functions is mediated by its binding to histone H4 and acetylation of H4K16 both in vivo and in vitro. Thus, our data assign a novel meiotic function to Esco2 beyond its role in the cohesion establishment during mouse oocyte meiosis.
A novel requirement for DROSHA in maintenance of mammalian CG methylation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-03 Athanasia Stathopoulou, Jyoti B. Chhetri, John C. Ambrose, Pierre-Olivier Estève, Lexiang Ji, Hediye Erdjument-Bromage, Guoqiang Zhang, Thomas A. Neubert, Sriharsa Pradhan, Javier Herrero, Robert J. Schmitz, Steen K.T. Ooi
In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem–loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.
Monitoring Replication Protein A (RPA) dynamics in homologous recombination through site-specific incorporation of non-canonical amino acids Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-12 Nilisha Pokhrel, Sofia Origanti, Eric Parker Davenport, Disha Gandhi, Kyle Kaniecki, Ryan A. Mehl, Eric C. Greene, Chris Dockendorff, Edwin Antony
An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance.
Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-14 Dekang Liu, Jane H. Frederiksen, Sascha E. Liberti, Anne Lützen, Guido Keijzers, Javier Pena-Diaz, Lene Juel Rasmussen
DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.
Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-24 Federica Bertoletti, Valentina Cea, Chih-Chao Liang, Taiba Lanati, Antonio Maffia, Mario D.M. Avarello, Lina Cipolla, Alan R. Lehmann, Martin A. Cohn, Simone Sabbioneda
DNA translesion synthesis (TLS) is a crucial damage tolerance pathway that oversees the completion of DNA replication in the presence of DNA damage. TLS polymerases are capable of bypassing a distorted template but they are generally considered inaccurate and they need to be tightly regulated. We have previously shown that polη is phosphorylated on Serine 601 after DNA damage and we have demonstrated that this modification is important for efficient damage bypass. Here we report that polη is also phosphorylated by CDK2, in the absence of damage, in a cell cycle-dependent manner and we identify serine 687 as an important residue targeted by the kinase. We discover that phosphorylation on serine 687 regulates the stability of the polymerase during the cell cycle, allowing it to accumulate in late S and G2 when productive TLS is critical for cell survival. Furthermore, we show that alongside the phosphorylation of S601, the phosphorylation of S687 and S510, S512 and/or S514 are important for damage bypass and cell survival after UV irradiation. Taken together our results provide new insights into how cells can, at different times, modulate DNA TLS for improved cell survival.
STN1–POLA2 interaction provides a basis for primase-pol α stimulation by human STN1 Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-19 Swapna Ganduri, Neal F. Lue
The CST (CTC1–STN1–TEN1) complex mediates critical functions in maintaining telomere DNA and overcoming genome-wide replication stress. A conserved biochemical function of the CST complex is its primase-Pol α (PP) stimulatory activity. In this report, we demonstrate the ability of purified human STN1 alone to promote PP activity in vitro. We show that this regulation is mediated primarily by the N-terminal OB fold of STN1, but does not require the DNA-binding activity of this domain. Rather, we observed a strong correlation between the PP-stimulatory activity of STN1 variants and their abilities to bind POLA2. Remarkably, the main binding target of STN1 in POLA2 is the latter's central OB fold domain. In the substrate-free structure of PP, this domain is positioned so as to block nucleic acid entry to the Pol α active site. Thus the STN1–POLA2 interaction may promote the necessary conformational change for nucleic acid delivery to Pol α and subsequent DNA synthesis. A disease-causing mutation in human STN1 engenders a selective defect in POLA2-binding and PP stimulation, indicating that these activities are critical for the in vivo function of STN1. Our findings have implications for the molecular mechanisms of PP, STN1 and STN1-related molecular pathology.
Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-29 David M. Wilson, Matthias Rieckher, Ashley B. Williams, Björn Schumacher
DNA interstrand crosslinks (ICLs) are generated by endogenous sources and chemotherapeutics, and pose a threat to genome stability and cell survival. Using Caenorhabditis elegans mutants, we identify DNA repair factors that protect against the genotoxicity of ICLs generated by trioxsalen/ultraviolet A (TMP/UVA) during development and aging. Mutations in nucleotide excision repair (NER) components (e.g. XPA-1 and XPF-1) imparted extreme sensitivity to TMP/UVA relative to wild-type animals, manifested as developmental arrest, defects in adult tissue morphology and functionality, and shortened lifespan. Compensatory roles for global-genome (XPC-1) and transcription-coupled (CSB-1) NER in ICL sensing were exposed. The analysis also revealed contributions of homologous recombination (BRC-1/BRCA1), the MUS-81, EXO-1, SLX-1 and FAN-1 nucleases, and the DOG-1 (FANCJ) helicase in ICL resolution, influenced by the replicative-status of the cell/tissue. No obvious or critical role in ICL repair was seen for non-homologous end-joining (cku-80) or base excision repair (nth-1, exo-3), the Fanconi-related proteins BRC-2 (BRCA2/FANCD1) and FCD-2 (FANCD2), the WRN-1 or HIM-6 (BLM) helicases, or the GEN-1 or MRT-1 (SNM1) nucleases. Our efforts uncover replication-dependent and -independent ICL repair networks, and establish nematodes as a model for investigating the repair and consequences of DNA crosslinks in metazoan development and in adult post-mitotic and proliferative germ cells.
A germline-limited piggyBac transposase gene is required for precise excision in Tetrahymena genome rearrangement Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-26 Lifang Feng, Guangying Wang, Eileen P. Hamilton, Jie Xiong, Guanxiong Yan, Kai Chen, Xiao Chen, Wen Dui, Amber Plemens, Lara Khadr, Arjune Dhanekula, Mina Juma, Hung Quang Dang, Geoffrey M. Kapler, Eduardo Orias, Wei Miao, Yifan Liu
Developmentally programmed genome rearrangement accompanies differentiation of the silent germline micronucleus into the transcriptionally active somatic macronucleus in the ciliated protozoan Tetrahymena thermophila. Internal eliminated sequences (IES) are excised, followed by rejoining of MAC-destined sequences, while fragmentation occurs at conserved chromosome breakage sequences, generating macronuclear chromosomes. Some macronuclear chromosomes, referred to as non-maintained chromosomes (NMC), are lost soon after differentiation. Large NMC contain genes implicated in development-specific roles. One such gene encodes the domesticated piggyBac transposase TPB6, required for heterochromatin-dependent precise excision of IES residing within exons of functionally important genes. These conserved exonic IES determine alternative transcription products in the developing macronucleus; some even contain free-standing genes. Examples of precise loss of some exonic IES in the micronucleus and retention of others in the macronucleus of related species suggest an evolutionary analogy to introns. Our results reveal that germline-limited sequences can encode genes with specific expression patterns and development-related functions, which may be a recurring theme in eukaryotic organisms experiencing programmed genome rearrangement during germline to soma differentiation.
Widespread intra-dependencies in the removal of introns from human transcripts Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-29 Seong Won Kim, Allison J. Taggart, Claire Heintzelman, Kamil J. Cygan, Caitlin G. Hull, Jing Wang, Barsha Shrestha, William G. Fairbrother
Research into the problem of splice site selection has followed a reductionist approach focused on how individual splice sites are recognized. Early applications of information theory uncovered an inconsistency. Human splice signals do not contain enough information to explain the observed fidelity of splicing. Here, we conclude that introns do not necessarily contain ‘missing’ information but rather may require definition from neighboring processing events. For example, there are known cases where an intronic mutation disrupts the splicing of not only the local intron but also adjacent introns. We present a genome-wide measurement of the order of splicing within human transcripts. The observed order of splicing cannot be explained by a simple kinetic model. Simulations reveal a bias toward a particular, transcript-specific order of intron removal in human genes. We validate an extreme class of intron that can only splice in a multi-intron context. Special categories of splicing such as exon circularization, first and last intron processing, alternative 5 and 3′ss usage and exon skipping are marked by distinct patterns of ordered intron removal. Excessive intronic length and silencer density tend to delay splicing. Shorter introns that contain enhancers splice early.
Regulation of HuR structure and function by dihydrotanshinone-I Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-18 Preet Lal, Linda Cerofolini, Vito Giuseppe D’Agostino, Chiara Zucal, Carmelo Fuccio, Isabelle Bonomo, Erik Dassi, Stefano Giuntini, Danilo Di Maio, Vikalp Vishwakarma, Ranjan Preet, Sha Neisha Williams, Max S. Fairlamb, Rachel Munk, Elin Lehrmann, Kotb Abdelmohsen, Saioa R. Elezgarai, Claudio Luchinat, Ettore Novellino, Alessandro Quattrone, Emiliano Biasini, Leonardo Manzoni, Myriam Gorospe, Dan A. Dixon, Pierfausto Seneci, Luciana Marinelli, Marco Fragai, Alessandro Provenzani
The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.
Antisense oligonucleotides targeting translation inhibitory elements in 5′ UTRs can selectively increase protein levels Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-21 Xue-hai Liang, Hong Sun, Wen Shen, Shiyu Wang, Joyee Yao, Michael T. Migawa, Huynh-Hoa Bui, Sagar S. Damle, Stan Riney, Mark J. Graham, Rosanne M. Crooke, Stanley T. Crooke
A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5′ UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5′ UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5′ UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.
Number of inadvertent RNA targets for morpholino knockdown in Danio rerio is largely underestimated: evidence from the study of Ser/Arg-rich splicing factors Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-21 Marine Joris, Marie Schloesser, Denis Baurain, Marc Hanikenne, Marc Muller, Patrick Motte
Although the involvement of Ser/Arg-rich (SR) proteins in RNA metabolism is well documented, their role in vertebrate development remains elusive. We, therefore, elected to take advantage of the zebrafish model organism to study the SR genes' functions using the splicing morpholino (sMO) microinjection and the programmable site-specific nucleases. Consistent with previous research, we revealed discrepancies between the mutant and morphant phenotypes and we show that these inconsistencies may result from a large number of unsuspected inadvertent morpholino RNA targets. While microinjection of MOs directed against srsf5a (sMOsrsf5a) led to developmental defects, the corresponding homozygous mutants did not display any phenotypic traits. Furthermore, microinjection of sMOsrsf5a into srsf5a−/− led to the previously observed morphant phenotype. Similar findings were observed for other SR genes. sMOsrsf5a alternative target genes were identified using deep mRNA sequencing. We uncovered that only 11 consecutive bases complementary to sMOsrsf5a are sufficient for binding and subsequent blocking of splice sites. In addition, we observed that sMOsrsf5a secondary targets can be reduced by increasing embryos growth temperature after microinjection. Our data contribute to the debate about MO specificity, efficacy and the number of unknown targeted sequences.
Concerted action of two 3′ cap-independent translation enhancers increases the competitive strength of translated viral genomes Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-24 Zhiyou Du, Olga M. Alekhina, Konstantin S. Vassilenko, Anne E. Simon
Several families of plant viruses evolved cap-independent translation enhancers (3′CITE) in the 3′ untranslated regions of their genomic (g)RNAs to compete with ongoing cap-dependent translation of cellular mRNAs. Umbravirus Pea enation mosaic virus (PEMV)2 is the only example where three 3′CITEs enhance translation: the eIF4E-binding Panicum mosaic virus-like translational enhancer (PTE) and ribosome-binding 3′ T-shaped structure (TSS) have been found in viruses of different genera, while the ribosome-binding kl-TSS that provides a long-distance interaction with the 5′ end is unique. We report that the PTE is the key translation promoting element, but inhibits translation in cis and in trans in the absence of the kl-TSS by sequestering initiation factor eIF4G. PEMV2 strongly outcompeted a cellular mRNA mimic for translation, indicating that the combination of kl-TSS and PTE is highly efficient. Transferring the 3′–5′ interaction from the kl-TSS to the PTE (to fulfill its functionality as found in other viruses) supported translationin vitro, but gRNA did not accumulate to detectable levels in protoplasts in the absence of the kl-TSS. It was shown that the PTE in conjunction with the kl-TSS did not markedly affect the translation initiation rate but rather increased the number of gRNAs available for translation. A model is proposed to explain how 3′CITE-based regulation of ribosome recruitment enhances virus fitness.
Co-produced natural ketolides methymycin and pikromycin inhibit bacterial growth by preventing synthesis of a limited number of proteins Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-29 Mashal M. Almutairi, Maxim S. Svetlov, Douglas A. Hansen, Nelli F. Khabibullina, Dorota Klepacki, Han-Young Kang, David H. Sherman, Nora Vázquez-Laslop, Yury S. Polikanov, Alexander S. Mankin
Antibiotics methymycin (MTM) and pikromycin (PKM), co-produced by Streptomyces venezuelae, represent minimalist macrolide protein synthesis inhibitors. Unlike other macrolides, which carry several side chains, a single desosamine sugar is attached to the macrolactone ring of MTM and PKM. In addition, the macrolactone scaffold of MTM is smaller than in other macrolides. The unusual structure of MTM and PKM and their simultaneous secretion by S. venezuelae bring about the possibility that two compounds would bind to distinct ribosomal sites. However, by combining genetic, biochemical and crystallographic studies, we demonstrate that MTM and PKM inhibit translation by binding to overlapping sites in the ribosomal exit tunnel. Strikingly, while MTM and PKM readily arrest the growth of bacteria, ∼40% of cellular proteins continue to be synthesized even at saturating concentrations of the drugs. Gel electrophoretic analysis shows that compared to other ribosomal antibiotics, MTM and PKM prevent synthesis of a smaller number of cellular polypeptides illustrating a unique mode of action of these antibiotics.
UbaLAI is a monomeric Type IIE restriction enzyme Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-26 Giedrius Sasnauskas, Giedrė Tamulaitienė, Gintautas Tamulaitis, Jelena Čalyševa, Miglė Laime, Renata Rimšelienė, Arvydas Lubys, Virginijus Siksnys
Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5′-CC/WGG-3′ (where W = A/T, and ‘/’ marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5′-CCWGG-3′ sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5′-CCWGG-3′ sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.
Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-26 Igor P. Oscorbin, Ekaterina A. Belousova, Ulyana A. Boyarskikh, Aleksandr I. Zakabunin, Evgeny A. Khrapov, Maksim L. Filipenko
At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants—urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.
Activities of gyrase and topoisomerase IV on positively supercoiled DNA Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-29 Rachel E. Ashley, Andrew Dittmore, Sylvia A. McPherson, Charles L. Turnbough, Keir C. Neuman, Neil Osheroff
Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ∼10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ∼3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.
The important conformational plasticity of DsrA sRNA for adapting multiple target regulation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-27 Pengzhi Wu, Xiaodan Liu, Lingna Yang, Yitong Sun, Qingguo Gong, Jihui Wu, Yunyu Shi
In bacteria, small non-coding RNAs (sRNAs) could function in gene regulations under variable stress responses. DsrA is an ∼90-nucleotide Hfq-dependent sRNA found in Escherichia coli. It regulates the translation and degradation of multiple mRNAs, such as rpoS, hns, mreB and rbsD mRNAs. However, its functional structure and particularly how it regulates multiple mRNAs remain obscure. Using NMR, we investigated the solution structures of the full-length and isolated stem–loops of DsrA. We first solved the NMR structure of the first stem–loop (SL1), and further studied the melting process of the SL1 induced by the base-pairing with the rpoS mRNA and the A-form duplex formation of the DsrA/rpoS complex. The secondary structure of the second stem–loop (SL2) was also determined, which contains a lower stem and an upper stem with distinctive stability. Interestingly, two conformational states of SL2 in dynamic equilibrium were observed in our NMR spectra, suggesting that the conformational selection may occur during the base-pairing between DsrA and mRNAs. In summary, our study suggests that the conformational plasticity of DsrA may represent a special mechanism sRNA employed to deal with its multiple regulatory targets of mRNA.
ALYREF mainly binds to the 5′ and the 3′ regions of the mRNA in vivo Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-10 Min Shi, Heng Zhang, Xudong Wu, Zhisong He, Lantian Wang, Shanye Yin, Bin Tian, Guohui Li, Hong Cheng
The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5′ end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3′ end of the mRNA. Furthermore, the 3′ processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5′ and the 3′ regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3′ processing.
hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-10 Naoko Kajitani, Jacob Glahder, Chengjun Wu, Haoran Yu, Kersti Nilsson, Stefan Schwartz
Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression.
The conserved AU dinucleotide at the 5′ end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-13 Chung-Shu Yeh, Shang-Lin Chang, Jui-Hui Chen, Hsuan-Kai Wang, Yue-Chang Chou, Chun-Hsiung Wang, Shih-Hsin Huang, Amy Larson, Jeffrey A Pleiss, Wei-Hau Chang, Tien-Hsien Chang
Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5′ splice site of a pre-mRNA and the 5′ end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5′-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5′ end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.
A structural map of oncomiR-1 at single-nucleotide resolution Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 Saikat Chakraborty, Yamuna Krishnan
The miR-17–92a cluster, also known as ‘oncomiR-1’, is an RNA transcript that plays a pivotal regulatory role in cellular processes, including the cell cycle, proliferation and apoptosis. Its dysregulation underlies the development of several cancers. Oncomir-1 comprises six constituent miRNAs, each processed with different efficiencies as a function of both developmental time and tissue type. The structural mechanisms that regulate such differential processing are unknown, and this has impeded our understanding of the dysregulation of oncomiR-1 in pathophysiology. By probing the sensitivity of each nucleotide in oncomiR-1 to reactive small molecules, we present a secondary structural map of this RNA at single-nucleotide resolution. The secondary structure and solvent accessible regions of oncomiR-1 reveal that most of its primary microRNA domains are suboptimal substrates for Drosha-DGCR8, and therefore resistant to microprocessing. The structure indicates that the binding of trans-acting factors is required to remodel the tertiary organization and unmask cryptic primary microRNA domains to facilitate their processing into pre-microRNAs.
Tuning RNA folding and function through rational design of junction topology Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 May Daher, Anthony M. Mustoe, Alex Morriss-Andrews, Charles L. Brooks III, Nils G. Walter
Structured RNAs such as ribozymes must fold into specific 3D structures to carry out their biological functions. While it is well-known that architectural features such as flexible junctions between helices help guide RNA tertiary folding, the mechanisms through which junctions influence folding remain poorly understood. We combine computational modeling with single molecule Förster resonance energy transfer (smFRET) and catalytic activity measurements to investigate the influence of junction design on the folding and function of the hairpin ribozyme. Coarse-grained simulations of a wide range of junction topologies indicate that differences in sterics and connectivity, independent of stacking, significantly affect tertiary folding and appear to largely explain previously observed variations in hairpin ribozyme stability. We further use our simulations to identify stabilizing modifications of non-optimal junction topologies, and experimentally validate that a three-way junction variant of the hairpin ribozyme can be stabilized by specific insertion of a short single-stranded linker. Combined, our multi-disciplinary study further reinforces that junction sterics and connectivity are important determinants of RNA folding, and demonstrates the potential of coarse-grained simulations as a tool for rationally tuning and optimizing RNA folding and function.
In vivo probing of nascent RNA structures reveals principles of cotranscriptional folding Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-14 Danny Incarnato, Edoardo Morandi, Francesca Anselmi, Lisa M. Simon, Giulia Basile, Salvatore Oliviero
Defining the in vivo folding pathway of cellular RNAs is essential to understand how they reach their final native conformation. We here introduce a novel method, named Structural Probing of Elongating Transcripts (SPET-seq), that permits single-base resolution analysis of transcription intermediates’ secondary structures on a transcriptome-wide scale, enabling base-resolution analysis of the RNA folding events. Our results suggest that cotranscriptional RNA folding in vivo is a mixture of cooperative folding events, in which local RNA secondary structure elements are formed as they get transcribed, and non-cooperative events, in which 5′-halves of long-range helices get sequestered into transient non-native interactions until their 3′ counterparts have been transcribed. Together our work provides the first transcriptome-scale overview of RNA cotranscriptional folding in a living organism.
Crystal structure of an engineered, HIV-specific recombinase for removal of integrated proviral DNA Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 Gretchen Meinke, Janet Karpinski, Frank Buchholz, Andrew Bohm
As part of the HIV infection cycle, viral DNA inserts into the genome of host cells such that the integrated DNA encoding the viral proteins is flanked by long terminal repeat (LTR) regions from the retrovirus. In an effort to develop novel genome editing techniques that safely excise HIV provirus from cells, Tre, an engineered version of Cre recombinase, was designed to target a 34-bp sequence within the HIV-1 LTR (loxLTR). The sequence targeted by Tre lacks the symmetry present in loxP, the natural DNA substrate for Cre. We report here the crystal structure of a catalytically inactive (Y324F) mutant of this engineered Tre recombinase in complex with the loxLTR DNA substrate. We also report that 17 of the 19 amino acid changes relative to Cre contribute to the altered specificity, even though many of these residues do not contact the DNA directly. We hypothesize that some mutations increase the flexibility of the Cre tetramer and that this, along with flexibility in the DNA, enable the engineered enzyme and DNA substrate to adopt complementary conformations.
A novel non-canonical PIP-box mediates PARG interaction with PCNA Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-12 Tanja Kaufmann, Irina Grishkovskaya, Anton A. Polyansky, Sebastian Kostrhon, Eva Kukolj, Karin M. Olek, Sebastien Herbert, Etienne Beltzung, Karl Mechtler, Thomas Peterbauer, Josef Gotzmann, Lijuan Zhang, Markus Hartl, Bojan Zagrovic, Kareem Elsayad, Kristina Djinovic-Carugo, Dea Slade
Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG–PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.
The fused SnoaL_2 domain in the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-18 Kapil Goutam, Arvind K. Gupta, Balasubramanian Gopal
Extra-cytoplasmic function (ECF) σ-factors are widespread in bacteria, linking environmental stimuli with changes in gene expression. These transcription factors span several phylogenetically distinct groups and are remarkably diverse in their activation and regulatory mechanisms. Here, we describe the structural and biochemical features of a Mycobacterium tuberculosis ECF factor σJ that suggests that the SnoaL_2 domain at the C-terminus can modulate the activity of this initiation factor in the absence of a cognate regulatory anti-σ factor. M. tuberculosis σJ can bind promoter DNA in vitro; this interaction is substantially impaired by the removal of the SnoaL_2 domain. This finding is consistent with assays to evaluate σJ-mediated gene expression. Structural similarity of the SnoaL_2 domain with epoxide hydrolases also suggests a novel functional role for this domain. The conserved sequence features between M. tuberculosis σJ and other members of the ECF41 family of σ-factors suggest that the regulatory mechanism involving the C-terminal SnoaL_2 domain is likely to be retained in this family of proteins. These studies suggest that the ECF41 family of σ-factors incorporate features of both—the σ70 family and bacterial one—component systems thereby providing a direct mechanism to implement environment-mediated transcription changes.
Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 Yurong Wen, Zhenlin Ouyang, Yue Yu, Xiaorong Zhou, Yingmei Pei, Bart Devreese, Paul G Higgins, Fang Zheng
AdeR–AdeS is a two-component regulatory system, which controls expression of the adeABC efflux pump involved in Acinetobacter baumannii multidrug resistance. AdeR is a response regulator consisting of an N-terminal receiver domain and a C-terminal DNA-binding-domain. AdeR binds to a direct-repeat DNA in the intercistronic region between adeR and adeABC. We demonstrate a markedly high affinity binding between unphosphorylated AdeR and DNA with a dissociation constant of 20 nM. In addition, we provide a 2.75 Å crystal structure of AdeR DNA-binding-domain complexed with the intercistronic DNA. This structure shows that the α3 and β hairpin formed by β5–β6 interacts with the major and minor groove of the DNA, which in turn leads to the introduction of a bend. The AdeR receiver domain structure revealed a dimerization motif mediated by a gearwheel-like structure involving the D108F109-R122 motif through cation π stack interaction. The structure of AdeR receiver domain bound with magnesium indicated a conserved Glu19Asp20-Asp63 magnesium-binding motif, and revealed that the potential phosphorylation site Asp63OD1 forms a hydrogen bond with Lys112. We thus dissected the mechanism of how AdeR recognizes the intercistronic DNA, which leads to a diverse mode of response regulation. Unlocking the AdeRS mechanism provides ways to circumvent A. baumannii antibiotic resistance.
Blocking the RecA activity and SOS-response in bacteria with a short α-helical peptide Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-02 Alexander Yakimov, Georgii Pobegalov, Irina Bakhlanova, Mikhail Khodorkovskii, Michael Petukhov, Dmitry Baitin
The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance.
Model-based design of RNA hybridization networks implemented in living cells Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-01 Guillermo Rodrigo, Satya Prakash, Shensi Shen, Eszter Majer, José-Antonio Daròs, Alfonso Jaramillo
Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs (natural or synthetic) are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermodynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations.
CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-18 Wallace H. Liu, Sarah C. Roemer, Alex M. Port, Mair E.A. Churchill
Nucleic Acids Res (2012) 40 (22): 11229–11239. doi :10.1093/nar/gks906
A novel requirement for DROSHA in maintenance of mammalian CG methylation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-17 Athanasia Stathopoulou, Jyoti B. Chhetri, John C. Ambrose, Pierre-Olivier Estève, Lexiang Ji, Hediye Erdjument-Bromage, Guoqiang Zhang, Thomas A. Neubert, Sriharsa Pradhan, Javier Herrero, Robert J. Schmitz, Steen K.T. Ooi
Nucleic Acids Res (2017). doi: 10.1093/nar/gkx695
Yeast CAF-1 assembles histone (H3-H4) 2 tetramers prior to DNA deposition Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-18 Duane D. Winkler, Hui Zhou, Mohd A. Dar, Zhiguo Zhang, Karolin Luger
Nucleic Acids Res 2012; 40 (20): 10139–10149. doi: 10.1093/nar/gks812
Combining asymmetric 13C-labeling and isotopic filter/edit NOESY: a novel strategy for rapid and logical RNA resonance assignment Nucleic Acids Res. (IF 10.162) Pub Date : 2017-08-09 Regan M. LeBlanc, Andrew P. Longhini, Stuart F.J. Le Grice, Bruce A. Johnson, Theodore K. Dayie
Although ∼98% of the human genomic output is transcribed as non-protein coding RNA, <2% of the protein data bank structures comprise RNA. This huge structural disparity stems from combined difficulties of crystallizing RNA for X-ray crystallography along with extensive chemical shift overlap and broadened linewidths associated with NMR of RNA. While half of the deposited RNA structures in the PDB were solved by NMR methods, the usefulness of NMR is still limited by the high cost of sample preparation and challenges of resonance assignment. Here we propose a novel strategy for resonance assignment that combines new strategic 13C labeling technologies with filter/edit type NOESY experiments to greatly reduce spectral complexity and crowding. This new strategy allowed us to assign important non-exchangeable resonances of proton and carbon (1′, 2′, 2, 5, 6 and 8) nuclei using only one sample and <24 h of NMR instrument time for a 27 nt model RNA. The method was further extended to assigning a 6 nt bulge from a 61 nt viral RNA element justifying its use for a wide range RNA chemical shift resonance assignment problems.
Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-13 Natascha Gödecke, Lisha Zha, Shawal Spencer, Sara Behme, Pamela Riemer, Michael Rehli, Hansjörg Hauser, Dagmar Wirth
Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-17 Shaked Afik, Kathleen B. Yates, Kevin Bi, Samuel Darko, Jernej Godec, Ulrike Gerdemann, Leo Swadling, Daniel C. Douek, Paul Klenerman, Eleanor J. Barnes, Arlene H. Sharpe, W. Nicholas Haining, Nir Yosef
The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV.
Detecting gene subnetworks under selection in biological pathways Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-18 Alexandre Gouy, Joséphine T. Daub, Laurent Excoffier
Advances in high throughput sequencing technologies have created a gap between data production and functional data analysis. Indeed, phenotypes result from interactions between numerous genes, but traditional methods treat loci independently, missing important knowledge brought by network-level emerging properties. Therefore, detecting selection acting on multiple genes affecting the evolution of complex traits remains challenging. In this context, gene network analysis provides a powerful framework to study the evolution of adaptive traits and facilitates the interpretation of genome-wide data. We developed a method to analyse gene networks that is suitable to evidence polygenic selection. The general idea is to search biological pathways for subnetworks of genes that directly interact with each other and that present unusual evolutionary features. Subnetwork search is a typical combinatorial optimization problem that we solve using a simulated annealing approach. We have applied our methodology to find signals of adaptation to high-altitude in human populations. We show that this adaptation has a clear polygenic basis and is influenced by many genetic components. Our approach, implemented in the R package signet, improves on gene-level classical tests for selection by identifying both new candidate genes and new biological processes involved in adaptation to altitude.
SupeRNAlign: a new tool for flexible superposition of homologous RNA structures and inference of accurate structure-based sequence alignments Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-20 Paweł Piątkowski, Jagoda Jabłońska, Adriana Żyła, Dorota Niedziałek, Dorota Matelska, Elżbieta Jankowska, Tomasz Waleń, Wayne K. Dawson, Janusz M. Bujnicki
RNA has been found to play an ever-increasing role in a variety of biological processes. The function of most non-coding RNA molecules depends on their structure. Comparing and classifying macromolecular 3D structures is of crucial importance for structure-based function inference and it is used in the characterization of functional motifs and in structure prediction by comparative modeling. However, compared to the numerous methods for protein structure superposition, there are few tools dedicated to the superimposing of RNA 3D structures. Here, we present SupeRNAlign (v1.3.1), a new method for flexible superposition of RNA 3D structures, and SupeRNAlign-Coffee—a workflow that combines SupeRNAlign with T-Coffee for inferring structure-based sequence alignments. The methods have been benchmarked with eight other methods for RNA structural superposition and alignment. The benchmark included 151 structures from 32 RNA families (with a total of 1734 pairwise superpositions). The accuracy of superpositions was assessed by comparing structure-based sequence alignments to the reference alignments from the Rfam database. SupeRNAlign and SupeRNAlign-Coffee achieved significantly higher scores than most of the benchmarked methods: SupeRNAlign generated the most accurate sequence alignments among the structure superposition methods, and SupeRNAlign-Coffee performed best among the sequence alignment methods.
De novo pathway-based biomarker identification Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-20 Nicolas Alcaraz, Markus List, Richa Batra, Fabio Vandin, Henrik J. Ditzel, Jan Baumbach
Gene expression profiles have been extensively discussed as an aid to guide the therapy by predicting disease outcome for the patients suffering from complex diseases, such as cancer. However, prediction models built upon single-gene (SG) features show poor stability and performance on independent datasets. Attempts to mitigate these drawbacks have led to the development of network-based approaches that integrate pathway information to produce meta-gene (MG) features. Also, MG approaches have only dealt with the two-class problem of good versus poor outcome prediction. Stratifying patients based on their molecular subtypes can provide a detailed view of the disease and lead to more personalized therapies. We propose and discuss a novel MG approach based on de novo pathways, which for the first time have been used as features in a multi-class setting to predict cancer subtypes. Comprehensive evaluation in a large cohort of breast cancer samples from The Cancer Genome Atlas (TCGA) revealed that MGs are considerably more stable than SG models, while also providing valuable insight into the cancer hallmarks that drive them. In addition, when tested on an independent benchmark non-TCGA dataset, MG features consistently outperformed SG models. We provide an easy-to-use web service at http://pathclass.compbio.sdu.dk where users can upload their own gene expression datasets from breast cancer studies and obtain the subtype predictions from all the classifiers.
The FASTK family of proteins: emerging regulators of mitochondrial RNA biology Nucleic Acids Res. (IF 10.162) Pub Date : 2017-09-19 Alexis A. Jourdain, Johannes Popow, Miguel A. de la Fuente, Jean-Claude Martinou, Paul Anderson, Maria Simarro
The FASTK family proteins have recently emerged as key post-transcriptional regulators of mitochondrial gene expression. FASTK, the founding member and its homologs FASTKD1–5 are architecturally related RNA-binding proteins, each having a different function in the regulation of mitochondrial RNA biology, from mRNA processing and maturation to ribosome assembly and translation. In this review, we outline the structure, evolution and function of these FASTK proteins and discuss the individual role that each has in mitochondrial RNA biology. In addition, we highlight the aspects of FASTK research that still require more attention.
cuRRBS: simple and robust evaluation of enzyme combinations for reduced representation approaches Nucleic Acids Res. (IF 10.162) Pub Date : 2017-09-19 Daniel E. Martin-Herranz, António J. M. Ribeiro, Felix Krueger, Janet M. Thornton, Wolf Reik, Thomas M. Stubbs
DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show in silico that cuRRBS-defined restriction enzymes consistently out-perform MspI digestion in many biological systems, considering both CpG and CHG contexts. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets.
Measurement of incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases using fluorescence microscopy Nucleic Acids Res. (IF 10.162) Pub Date : 2017-09-19 Matthew T. Walsh, Xiaohua Huang
We describe a method for measuring the single-turnover incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases. Time-lapse total internal reflection fluorescence (TIRF) microscopy is used to directly measure the kinetics of single-base nucleotide incorporation into primed DNA templates covalently attached to the surface of a glass coverslip using a fixed ratio of a native nucleotide and a corresponding fluorescently labeled nucleotide over a series of total nucleotide concentrations. The presence of a labeled nucleotide allows for the kinetics of competitive incorporation reactions by DNA polymerase to be monitored. The single-turnover catalytic rate constants and Michaelis constants of the incorporation of the native nucleotides can be determined by modeling the kinetics of the parallel competitive reactions. Our method enables the measurements of the kinetics parameters of incorporation of native or other non-fluorescent nucleotides without using a rapid stopped-flow or quench-flow instrument and the generally more involved and less quantitative post-reaction analysis of the reaction products. As a demonstration of our method, we systematically determined the single-turnover incorporation kinetics of all four native nucleotides and a set of Cy3-labeled nucleotides by the Klenow fragment of Escherichia coli DNA polymerase I.
Detecting and characterizing microRNAs of diverse genomic origins via miRvial Nucleic Acids Res. (IF 10.162) Pub Date : 2017-09-19 Jing Xia, Lun Li, Tiantian Li, Zhiwei Fang, Kevin Zhang, Junfei Zhou, Hai Peng, Weixiong Zhang
MicroRNAs form an essential class of post-transcriptional gene regulator of eukaryotic species, and play critical parts in development and disease and stress responses. MicroRNAs may originate from various genomic loci, have structural characteristics, and appear in canonical or modified forms, making them subtle to detect and analyze. We present miRvial, a robust computational method and companion software package that supports parameter adjustment and visual inspection of candidate microRNAs. Extensive results comparing miRvial and six existing microRNA finding methods on six model organisms, Mus musculus, Drosophila melanogaste, Arabidopsis thaliana, Oryza sativa, Physcomitrella patens and Chlamydomonas reinhardtii, demonstrated the utility and rigor of miRvial in detecting novel microRNAs and characterizing features of microRNAs. Experimental validation of several novel microRNAs in C. reinhardtii that were predicted by miRvial but missed by the other methods illustrated the superior performance of miRvial over the existing methods. miRvial is open source and available at https://github.com/SystemsBiologyOfJianghanUniversity/miRvial.
iSyTE 2.0: a database for expression-based gene discovery in the eye Nucleic Acids Res. (IF 10.162) Pub Date : 2017-09-19 Atul Kakrana, Andrian Yang, Deepti Anand, Djordje Djordjevic, Deepti Ramachandruni, Abhyudai Singh, Hongzhan Huang, Joshua W. K. Ho, Salil A. Lachke
Although successful in identifying new cataract-linked genes, the previous version of the database iSyTE (integrated Systems Tool for Eye gene discovery) was based on expression information on just three mouse lens stages and was functionally limited to visualization by only UCSC-Genome Browser tracks. To increase its efficacy, here we provide an enhanced iSyTE version 2.0 (URL: http://research.bioinformatics.udel.edu/iSyTE) based on well-curated, comprehensive genome-level lens expression data as a one-stop portal for the effective visualization and analysis of candidate genes in lens development and disease. iSyTE 2.0 includes all publicly available lens Affymetrix and Illumina microarray datasets representing a broad range of embryonic and postnatal stages from wild-type and specific gene-perturbation mouse mutants with eye defects. Further, we developed a new user-friendly web interface for direct access and cogent visualization of the curated expression data, which supports convenient searches and a range of downstream analyses. The utility of these new iSyTE 2.0 features is illustrated through examples of established genes associated with lens development and pathobiology, which serve as tutorials for its application by the end-user. iSyTE 2.0 will facilitate the prioritization of eye development and disease-linked candidate genes in studies involving transcriptomics or next-generation sequencing data, linkage analysis and GWAS approaches.
A snoRNA modulates mRNA 3′ end processing and regulates the expression of a subset of mRNAs Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-26 Chunliu Huang, Junjie Shi, Yibin Guo, Weijun Huang, Shanshan Huang, Siqi Ming, Xingui Wu, Rui Zhang, Junjun Ding, Wei Zhao, Jie Jia, Xi Huang, Andy Peng Xiang, Yongsheng Shi, Chengguo Yao
mRNA 3′ end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3′ processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3′ processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3′ processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1–RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3′ processing.
mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation Nucleic Acids Res. (IF 10.162) Pub Date : 2017-06-28 Anna M. Rydzik, Marcin Warminski, Pawel J. Sikorski, Marek R. Baranowski, Sylwia Walczak, Joanna Kowalska, Joanna Zuberek, Maciej Lukaszewicz, Elzbieta Nowak, Timothy D. W. Claridge, Edward Darzynkiewicz, Marcin Nowotny, Jacek Jemielity
Analogues of the mRNA 5′-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.
Selective tumor cell death induced by irradiated riboflavin through recognizing DNA G–T mismatch Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-12 Yi Yuan, Yongyun Zhao, Lianqi Chen, Jiasi Wu, Gangyi Chen, Sheng Li, Jiawei Zou, Rong Chen, Jian Wang, Fan Jiang, Zhuo Tang
Riboflavin (vitamin B2) has been thought to be a promising antitumoral agent in photodynamic therapy, though the further application of the method was limited by the unclear molecular mechanism. Our work reveals that riboflavin was able to recognize G–T mismatch specifically and induce single-strand breaks in duplex DNA targets efficiently under irradiation. In the presence of riboflavin, the photo-irradiation could induce the death of tumor cells that are defective in mismatch repair system selectively, highlighting the G–T mismatch as potential drug target for tumor cells. Moreover, riboflavin is a promising leading compound for further drug design due to its inherent specific recognition of the G–T mismatch.
Multimerization rules for G-quadruplexes Nucleic Acids Res. (IF 10.162) Pub Date : 2017-07-24 Sofia Kolesnikova, Martin Hubálek, Lucie Bednárová, Josef Cvačka, Edward A. Curtis
G-quadruplexes can multimerize under certain conditions, but the sequence requirements of such structures are not well understood. In this study, we investigated the ability of all possible variants of the central tetrad in a monomeric, parallel-strand G-quadruplex to form higher-order structures. Although most of these 256 variants existed primarily as monomers under the conditions of our screen, ∼10% formed dimers or tetramers. These structures could form in a wide range of monovalent and divalent metal ions, and folding was highly cooperative in both KCl and MgCl2. As was previously shown for G-quadruplexes that bind GTP and promote peroxidase reactions, G-quadruplexes that form dimers and tetramers have distinct sequence requirements. Some mutants could also form heteromultimers, and a second screen was performed to characterize the sequence requirements of these structures. Taken together, these experiments provide new insights into the sequence requirements and structures of both homomultimeric and heteromultimeric G-quadruplexes.
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