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https://www.readbyqxmd.com/read/28434903/measuring-transcription-dynamics-in-living-cells-using-a-photobleaching-approach
#1
REVIEW
Hodaya Hochberg, Yehuda Brody, Yaron Shav-Tal
The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics...
April 20, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28433859/photoelectrochemical-immunosensor-for-methylated-rna-detection-based-on-g-c3n4-cds-quantum-dots-heterojunction-and-phos-tag-biotin
#2
Haiyan Wang, Qihai Zhang, Huanshun Yin, Minghui Wang, Wenjing Jiang, Shiyun Ai
N(6)-methyladenosine (m(6)A) is an enigmatic and abundant internal modification in eukaryotic messenger RNA (mRNA), which could affect various aspects of RNA metabolism and mRNA translation. Herein, a novel photoelectrochemical (PEC) immunosensor was constructed for m(6)A detection based on the inhibition of Cu(2+) to the photoactivity of g-C3N4/CdS quantum dots (g-C3N4/CdS) heterojunction, where g-C3N4/CdS heterojunction was used as photoactive material, anti-m(6)A antibody as recognition unit for m(6)A-containing RNA, Phos-tag-biotin as link unit and avidin functionalized CuO as PEC signal indicator...
April 12, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28416596/microrna-profiling-reveals-marker-of-motor-neuron-disease-in-als-models
#3
Mariah L Hoye, Erica D Koval, Amy J Wegener, Theodore S Hyman, Chengran Yang, David R O'Brien, Rebecca L Miller, Tracy Cole, Kathleen M Schoch, Tao Shen, Tomonori Kunikata, Jean-Philippe Richard, David H Gutmann, Nicholas J Maragakis, Holly B Kordasiewicz, Joseph D Dougherty, Timothy M Miller
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type in order to elucidate underlying disease mechanisms and identify novel targets that inform on MN health during ALS disease timecourse. microRNAs (miRNAs) are short, non-coding RNAs that can shape the expression profile of a cell and, consequently, often exhibit cell type enriched expression...
April 17, 2017: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
https://www.readbyqxmd.com/read/28413437/gostag-gene-ontology-subtrees-to-tag-and-annotate-genes-within-a-set
#4
Brian D Bennett, Pierre R Bushel
BACKGROUND: Over-representation analysis (ORA) detects enrichment of genes within biological categories. Gene Ontology (GO) domains are commonly used for gene/gene-product annotation. When ORA is employed, often times there are hundreds of statistically significant GO terms per gene set. Comparing enriched categories between a large number of analyses and identifying the term within the GO hierarchy with the most connections is challenging. Furthermore, ascertaining biological themes representative of the samples can be highly subjective from the interpretation of the enriched categories...
2017: Source Code for Biology and Medicine
https://www.readbyqxmd.com/read/28406973/tre5-a-retrotransposition-profiling-reveals-putative-rna-polymerase-iii-transcription-complex-binding-sites-on-the-dictyostelium-extrachromosomal-rdna-element
#5
Thomas Spaller, Marco Groth, Gernot Glöckner, Thomas Winckler
The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes...
2017: PloS One
https://www.readbyqxmd.com/read/28400296/recombinant-expression-and-purification-of-the-rna-binding-larp6-proteins-from-fish-genetic-model-organisms
#6
José M Castro, Daniel A Horn, Karen A Lewis
The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His6-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components...
April 8, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28399446/enzyme-mediated-tagging-of-rna
#7
REVIEW
Lea Anhäuser, Andrea Rentmeister
RNA molecules can play diverse roles in the cell owing to their secondary structure dynamics and various binding modes. Studying localization and dynamics of RNA in vitro or in cells requires tagging with suitable reporter molecules-fluorophores being the most prominent ones. Enzymatic RNA labeling approaches are currently emerging as valuable alternatives to purely chemical synthesis and to binding- or hybridization-based RNA-imaging approaches. Different classes of enzymes allow for cotranscriptional or posttranscriptional installation of small functional groups in RNA...
April 8, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/28396352/association-of-the-cold-shock-dead-box-rna-helicase-rhle-to-the-rna-degradosome-in-caulobacter-crescentus
#8
Angel A Aguirre, Alexandre M Vicente, Steven W Hardwick, Daniela M Alvelos, Ricardo R Mazzon, Ben F Luisi, Marilis V Marques
In diverse bacterial lineages, multi-enzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic, Gram-negative bacteria Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below the optimum for growth...
April 10, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28392367/six-gu-rich-6gur-fus-binding-motifs-detected-by-normalization-of-clip-seq-by-nascent-seq
#9
Jun-Ichi Takeda, Akio Masuda, Kinji Ohno
FUS, an RNA-binding protein (RBP), is mutated or abnormally regulated in neurodegenerative disorders. FUS regulates various aspects of RNA metabolisms. FUS-binding sites are rich in GU contents and are highly degenerative. FUS-binding motifs of GGU, GGUG, GUGGU and CGCGC have been previously reported. These motifs, however, are applicable to a small fraction of FUS-binding sites. As CLIP-seq tags are enriched in genes that are highly expressed, we normalized CLIP-seq tags by Nascent-seq tags or RNA-seq tags of mouse N2a cells...
April 6, 2017: Gene
https://www.readbyqxmd.com/read/28388938/subtyping-of-blastocystis-sp-isolated-from-symptomatic-and-asymptomatic-individuals-in-makkah-saudi-arabia
#10
Raafat T Mohamed, Mohammed A El-Bali, Anhar A Mohamed, Mona A Abdel-Fatah, Mohamed A El-Malky, Nawras M Mowafy, Dina A Zaghlool, Rowaida A Bakri, Saeed A Al-Harthi
BACKGROUND: Blastocystis is a group of cosmopolitan gastrointestinal parasite of humans and a wide variety of animals. These anaerobic protozoans include more than 17 specific small-subunit ribosomal RNA subtypes, of which nine are found in humans with a variable geographical distribution. Until now, no study has described the Blastocystis subtypes present in Saudi Arabia. METHODS: In total, 1,262 faecal samples were collected from patients with gastrointestinal complaints and asymptomatic individuals visiting two major hospitals...
April 7, 2017: Parasites & Vectors
https://www.readbyqxmd.com/read/28377111/genome-wide-identification-of-micrornas-involved-in-fatty-acid-and-lipid-metabolism-of-brassica-napus-by-small-rna-and-degradome-sequencing
#11
Zhiwei Wang, Yan Qiao, Jingjing Zhang, Wenhui Shi, Jinwen Zhang
Rapeseed (Brassica napus) is an important cash crop considered as the third largest oil crop worldwide. Rapeseed oil contains various saturation or unsaturation fatty acids, these fatty acids, whose could incorporation with TAG form into lipids stored in seeds play various roles in the metabolic activity. The different fatty acids in B. napus seeds determine oil quality, define if the oil is edible or must be used as industrial material. miRNAs are kind of non-coding sRNAs that could regulate gene expressions through post-transcriptional modification to their target transcripts playing important roles in plant metabolic activities...
April 1, 2017: Gene
https://www.readbyqxmd.com/read/28367472/hiwi2-rs508485-polymorphism-is-associated-with-non-obstructive-azoospermia-in-iranian-patients
#12
Zeeba Kamaliyan, Sara Pouriamanesh, Mona Amin-Beidokhti, Amir Rezagholizadeh, Reza Mirfakhraie
BACKGROUND: The PIWI-interacting RNA (piRNA) pathway has an essential role in transposon silencing, meiosis progression, spermatogenesis, and germline maintenance. HIWI genes are critical for piRNA biogenesis and function. Therefore, polymorphisms in HIWI genes contribute to spermatogenesis defects and can be considered as risk factors for male infertility. The aim of the present study was to investigate the association between the HIWI2 gene rs508485 polymorphism and non-obstructive azoospermia...
April 2017: Reports of Biochemistry & Molecular Biology
https://www.readbyqxmd.com/read/28364023/rna-activation-independent-dna-targeting-of-the-type-iii-crispr-cas-system-by-a-csm-complex
#13
Kwang-Hyun Park, Yan An, Tae-Yang Jung, In-Young Baek, Haemin Noh, Woo-Chan Ahn, Hans Hebert, Ji-Joon Song, Jeong-Hoon Kim, Byung-Ha Oh, Eui-Jeon Woo
The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1121334151, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities...
March 31, 2017: EMBO Reports
https://www.readbyqxmd.com/read/28362374/a-g-quadruplex-dna-affinity-approach-for-purification-of-enzymatically-active-g4-resolvase1
#14
Eric D Routh, Steven D Creacy, Peter E Beerbower, Steven A Akman, James P Vaughn, Philip J Smaldino
Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al...
March 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28360904/host-factors-in-the-infection-cycle-of-bamboo-mosaic-virus
#15
REVIEW
Ying-Ping Huang, I-Hsuan Chen, Ching-Hsiu Tsai
To complete the infection cycle efficiently, the virus must hijack the host systems in order to benefit for all the steps and has to face all the defense mechanisms from the host. This review involves a discussion of how these positive and negative factors regulate the viral RNA accumulation identified for the Bamboo mosaic virus (BaMV), a single-stranded RNA virus. The genome of BaMV is approximately 6.4 kb in length, encoding five functional polypeptides. To reveal the host factors involved in the infection cycle of BaMV, a few different approaches were taken to screen the candidates...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28356503/exo70c2-is-a-key-regulatory-factor-for-optimal-tip-growth-pollen
#16
Lukas Synek, Nemanja Vukasinovic, Ivan Kulich, Michal Hala, Klara Aldorfova, Matyas Fendrych, Viktor Zarsky
The exocyst, an eukaryotic tethering complex, co-regulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, C1, C2, F1, H3, H5, and H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth...
March 29, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28351991/the-composition-of-the-arabidopsis-rna-polymerase-ii-transcript-elongation-complex-reveals-the-interplay-between-elongation-and-mrna-processing-factors
#17
Wojciech Antosz, Alexander Pfab, Hans F Ehrnsberger, Philipp Holzinger, Karin Köllen, Simon A Mortensen, Astrid Bruckmann, Thomas Schubert, Gernot Längst, Joachim Griesenbeck, Veit Schubert, Marion Grasser, Klaus D Grasser
Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity-purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana, the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS co-purified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodelling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs...
March 28, 2017: Plant Cell
https://www.readbyqxmd.com/read/28349467/crosslinking-methods-to-identify-rna-methyltransferase-targets-in-vivo
#18
Sara Haag, Jens Kretschmer, Katherine E Sloan, Markus T Bohnsack
Several crosslinking methods have been developed to identify interacting RNAs for proteins of interest. Here, we describe variants of the UV crosslinking and analysis of cDNA (CRAC) method that allow target identification of RNA methyltransferases on a genome-wide scale. We present a detailed protocol for the application of CRAC in human cells that stably express the protein of interest fused to a tandem affinity tag. After the introduction of a covalent link between the protein and its target RNAs, protein-RNA complexes are purified and bound RNAs trimmed, ligated to adapters, reverse transcribed, and amplified...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28341446/pkr-induces-the-expression-of-nlrp3-by-regulating-the-nf-%C3%AE%C2%BAb-pathway-in-porphyromonas-gingivalis-infected-osteoblasts
#19
Kaya Yoshida, Hirohiko Okamura, Yuka Hiroshima, Kaori Abe, Jun-Ichi Kido, Yasuo Shinohara, Kazumi Ozaki
The double-stranded RNA-dependent kinase (PKR), which is activated by double stranded RNA, induces inflammation by regulating NF-κB signaling. The NLR family pyrin domain-containing 3 (NLRP3) inflammasome also modulates inflammation in response to infection. Porphyromonas gingivalis (P.gingivalis) is an oral bacterium which is implicated in the pathogenesis of periodontal diseases. We previously reported that PKR is a key modulator of bone metabolism and inflammation in the periodontal tissue. PKR was also reported to induce inflammation in response to microbes by regulating the NLRP3 inflammasome, suggesting that PKR could affect inflammation along with NLRP3 in periodontal diseases...
March 22, 2017: Experimental Cell Research
https://www.readbyqxmd.com/read/28337393/development-and-validation-of-est-ssr-markers-for-fokienia-hodginsii-cupressaceae
#20
Mingyan Ding, Kaikai Meng, Qiang Fan, Weizheng Tan, Wenbo Liao, Sufang Chen
PREMISE OF THE STUDY: Fokienia hodginsii (Cupressaceae) is a Tertiary relict evergreen conifer of the monotypic genus Fokienia. Polymorphic microsatellite markers were developed to investigate its genetic diversity and population structure. METHODS AND RESULTS: RNA transcripts of F. hodginsii were sequenced and de novo assembled into 85,818 unigenes, and 1892 simple sequence repeat (SSR) markers were detected from the unigenes. A total of 273 expressed sequence tag-SSR primer pairs were designed and tested, and 129 successfully amplified...
March 2017: Applications in Plant Sciences
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