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RNA tagging

Manisha B Walunj, Arun A Tanpure, Seergazhi G Srivatsan
Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction...
March 13, 2018: Nucleic Acids Research
Neftali Vazquez, Lilia Sanchez, Rebecca Marks, Eduardo Martinez, Victor Fanniel, Alma Lopez, Andrea Salinas, Itzel Flores, Jesse Hirschmann, Robert Gilkerson, Erin Schuenzel, Robert Dearth, Reginald Halaby, Wendy Innis-Whitehouse, Megan Keniry
BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone...
March 14, 2018: BMC Molecular Biology
Dieter Galea, Ivan Laponogov, Kirill Veselkov
Motivation: Recognition of biomedical entities from scientific text is a critical component of natural language processing and automated information extraction platforms. Modern named entity recognition approaches rely heavily on supervised machine learning techniques, which are critically dependent on annotated training corpora. These approaches have been shown to perform well when trained and tested on the same source. However, in such scenario, the performance and evaluation of these models may be optimistic, as such models may not necessarily generalize to independent corpora, resulting in potential non-optimal entity recognition for large-scale tagging of widely diverse articles in databases such as PubMed...
March 10, 2018: Bioinformatics
Hernan Garcia-Ruiz, Sergio M Gabriel Peralta, Patricia A Harte-Maxwell
Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro)...
March 14, 2018: Viruses
Yves Mugabo, Mina Sadeghi, Nancy N Fang, Thibault Mayor, Gareth E Lim
Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a TAP-epitope-tagged 14-3-3ζ molecule...
March 12, 2018: Journal of Biological Chemistry
Miaomiao Xing, Chao Sun, Hailong Li, Shilin Hu, Lei Lei, Jungen Kang
Cabbage (Brassica oleracea L. var. capitata), an important vegetable crop in the Brassicaceae family, is economically important worldwide. In the process of hybrid seed production, Ogura cytoplasmic male sterility (OguCMS), controlled by the mitochondrial gene orf138, has been extensively used for cabbage hybrid production with complete and stable male sterility. To identify the critical genes and pathways involved in the sterility and to better understand the underlying molecular mechanisms, the anther of OguCMS line R2P2CMS and the fertile line R2P2 were used for RNA-seq and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) proteome analysis...
2018: PloS One
Laura L Burger, Charlotte Vanacker, Chayarndorn Phumsatitpong, Elizabeth R Wagenmaker, Luhong Wang, David P Olson, Suzanne M Moenter
GnRH neurons are a nexus of fertility regulation. We utilized translating ribosome affinity purification coupled with RNA sequencing to examine mRNAs of GnRH neurons in adult intact and gonadectomized (GDX) male and female mice. GnRH neuron ribosomes were tagged with green fluorescent protein (GFP) and GFP-labeled polysomes isolated by immunoprecipitation, producing one RNA fraction enhanced for GnRH neuron transcripts and one RNA fraction depleted. cDNA libraries were created from each fraction and 50-base, paired-end sequencing done and differential expression (enhanced fraction/depleted fraction) determined with a threshold of >1...
March 7, 2018: Endocrinology
Piyush K Jain, Simon H Friedman
There is a need for methods to chemically incorporate photocleavable labels into synthetic and biologically sourced nucleic acids in a chemically defined and reversible manner. We have previously demonstrated that the light cleaved diazo-DMNPE group (diazo di-methoxy nitro phenyl ethyl) has a remarkable regiospecificity for modifying terminally phosphorylated siRNA. Building on this observation, we have identified conditions by which a new diazo-DMNPE reagent (diazo-DMNPE-azide or DDA) is able to singly modify any nucleic acid (RNA, DNA, single stranded, double stranded, 3' or 5' phosphate)...
March 7, 2018: Chembiochem: a European Journal of Chemical Biology
V Siddartha Yerramilli, Kyung Hyuk Kim
RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells...
March 7, 2018: ACS Synthetic Biology
Hoan T Ngo, Elizabeth Freedman, Ren Abelard Odion, Pietro Strobbia, Agampodi Swarnapali De Silva Indrasekara, Priya Vohra, Steve M Taylor, Tuan Vo-Dinh
Direct detection of genetic biomarkers in body fluid lysate without target amplification will revolutionize nucleic acid-based diagnostics. However, the low concentration of target sequences makes this goal challenging. We report a method for direct detection of pathogen RNA in blood lysate using a bioassay using surface-enhanced Raman spectroscopy (SERS)-based detection integrated in a "lab-in-a-stick" portable device. Two levels of signal enhancement were employed to achieve the sensitivity required for direct detection...
March 6, 2018: Scientific Reports
Ayaka Ito, Chieko Sugita, Mizuho Ichinose, Yoshinobu Kato, Hiroshi Yamamoto, Toshiharu Shikanai, Mamoru Sugita
Pentatricopeptide repeat (PPR) proteins are known to play important roles in posttranscriptional regulation in plant organelles. However, the function of the majority of PPR proteins remains unknown. To examine their functions, Physcomitrella patens PpPPR_66 knockout (KO) mutants were generated and characterized. KO mosses exhibited a wild type-like growth phenotype but showed aberrant chlorophyll fluorescence due to defects in chloroplast NADH dehydrogenase-like (NDH) activity. Immuno-blot analysis suggested that disruption of PpPPR_66 led to a complete loss of the chloroplast NDH complex...
March 5, 2018: Plant Journal: for Cell and Molecular Biology
Martina Dvořáčková, Jiří Fajkus
Thanks to recent innovative methodologies, key cellular processes such as replication or transcription can be visualized directly in situ in intact tissues. Many studies use so-called click iT chemistry where nascent DNA can be tracked by 5-ethynyl-2'-deoxyuridine (EdU), and nascent RNA by 5-ethynyl uridine (EU). While the labeling of replicating DNA by EdU has already been well established and further exploited in plants, the use of EU to reveal nascent RNA has not been developed to such an extent. In this article, we present a protocol for labeling of nucleolar RNA transcripts using EU and show that EU effectively highlights the nucleolus...
2018: Frontiers in Plant Science
Yaling Yu, Haomin Cui, Chuan Chen, Gen Wen, Jia Xu, Binbin Zheng, Jianse Zhang, Chunyang Wang, Yimin Chai, Jin Mei
Although mammalian kidney regeneration has been reported to occur throughout life, mature kidneys in mammals are not thought to regenerate sufficiently, particularly glomeruli. In our previous work, we found that renal regeneration could be enhanced by decellularized renal scaffolds after partial nephrectomy. In this study, we verified that the enhanced renal regeneration mediated by decellularized scaffolds could be attributed to regenerated glomeruli, which were counted both indirectly and directly under a microscope...
February 24, 2018: Biomaterials
Diana Bauermeister, Tomas Pieler
Whole-mount in situ hybridization (WMISH) is a common approach that is used to visualize spatial and temporal gene expression in embryos. In this process, digoxygenin-labeled antisense RNA is hybridized to the complementary transcript of interest and RNA hybrids are immunohistochemically detected using an alkaline phosphatase-conjugated antibody against digoxigenin. During Xenopus laevis oogenesis, certain RNAs localize to the animal or vegetal pole laying the foundation for germ cell development and germ layer formation of the future embryo...
March 1, 2018: Cold Spring Harbor Protocols
Ying Wang, Jin-Jun Cao, Wei-Hai Li
Stoneflies comprise an ancient group of insects, but the phylogenetic position of Plecoptera and phylogenetic relations within Plecoptera have long been controversial, and more molecular data is required to reconstruct precise phylogeny. Herein, we present the complete mitogenome of a stonefly, Suwallia teleckojensis , which is 16146 bp in length and consists of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and a control region (CR). Most PCGs initiate with the standard start codon ATN...
February 28, 2018: International Journal of Molecular Sciences
Xiaoying Liang, Kevin J Hart, Gang Dong, Faiza A Siddiqui, Aswathy Sebastian, Xiaolian Li, Istvan Albert, Jun Miao, Scott E Lindner, Liwang Cui
In this study, we characterized a Puf family gene member, Puf3, in the malaria parasites Plasmodium falciparum and Plasmodium yoelii Secondary structure prediction suggested that the RNA-binding domains of Puf3s consisted of 11 pumilio repeats, similar to those in human Puf-A and yeast Puf6, which are involved in ribosome biogenesis. Neither pfpuf3 nor pypuf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle. Cellular fractionation of PfPuf3 revealed preferential partitioning to the nuclear fraction, consistent with nuclear localization of PfPuf3::GFP and PyPuf3::GFP detected by immunofluorescence...
February 27, 2018: Journal of Cell Science
Marta Robledo, Ana M Matia-González, Natalia I García-Tomsig, José I Jiménez-Zurdo
The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin...
2018: Methods in Molecular Biology
Jonathan Gans, Jonathan Osborne, Juliet Cheng, Louise Djapgne, Amanda G Oglesby-Sherrouse
Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules...
2018: Methods in Molecular Biology
Marie-Claude Carrier, Guillaume Laliberté, Eric Massé
Small regulatory RNAs (sRNAs) are ubiquitous regulatory molecules expressed in living cells. In prokaryotes, sRNAs usually bind to target mRNAs to either promote their degradation or interfere with translation initiation. Because a single sRNA can regulate a considerable number of target mRNAs, we seek to identify those targets rapidly and reliably. Here, we present a robust method based on the co-purification of target mRNAs bound to MS2-tagged sRNAs expressed in vivo. After purification of the tagged-sRNA, we use RNAseq to determine the identity of all RNA interacting partners and their enrichment level...
2018: Methods in Molecular Biology
Toshitsugu Fujita, Miyuki Yuno, Hodaka Fujii
OBJECTIVE: Previously, we developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology, which isolates specific genomic regions while preserving their molecular interactions. In enChIP, the locus of interest is tagged with engineered DNA-binding molecules such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically inactive form of Cas9 (dCas9) and guide RNA, followed by affinity purification of the tagged locus to allow identification of associated molecules...
February 27, 2018: BMC Research Notes
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