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mRNA decay

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https://www.readbyqxmd.com/read/29236262/studying-nonsense-mediated-mrna-decay-in-mammalian-cells-using-a-multicolored-bioluminescence-based-reporter-system
#1
Andrew Nickless, Zhongsheng You
The nonsense-mediated mRNA decay (NMD) pathway degrades aberrant transcripts containing premature translation termination codons (PTCs) and also regulates the levels of many normal mRNAs containing NMD-inducing features. The activity of this pathway varies considerably in different cell types and can change in response to developmental and environmental cues. Modulating NMD activity represents a potential therapeutic avenue for certain genetic disorders and cancers. Simple reporter systems capable of faithfully assessing NMD activity in mammalian cells greatly facilitate both basic and translational research on NMD...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236261/reactivation-assay-to-identify-direct-targets-of-the-nonsense-mediated-mrna-decay-pathway-in-drosophila
#2
Jonathan O Nelson, Mark M Metzstein
Transcriptome analysis provides a snapshot of cellular gene expression and is used to determine how cells and organisms respond to genetic or environmental changes. Identifying the transcripts whose expression levels are regulated directly by the manipulation being examined from those whose expression changes as a secondary cause from the primary changes requires additional analyses. Here we present a technique used to distinguish direct targets of the nonsense-mediated mRNA decay (NMD) pathway in Drosophila from secondary gene expression effects caused by loss of this pathway...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236260/generation-of-cell-lines-stably-expressing-a-fluorescent-reporter-of-nonsense-mediated-mrna-decay-activity
#3
Nadezhda M Markina, Anton P Pereverzev, Dmitry B Staroverov, Konstantin A Lukyanov, Nadya G Gurskaya
Nonsense-mediated mRNA decay (NMD) is a mechanism of mRNA surveillance ubiquitous among eukaryotes. Importantly, NMD not only removes aberrant transcripts with premature stop codons, but also regulates expression of many normal genes. A recently introduced dual-color fluorescent protein-based reporter enables analysis of NMD activity in live cells. In this chapter we describe the method to generate stable transgenic cell lines expressing the splicing-dependent NMD reporter using consecutive steps of lentivirus transduction and Tol2 transposition...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236259/identifying-cellular-nonsense-mediated-mrna-decay-nmd-targets-immunoprecipitation-of-phosphorylated-upf1-followed-by-rna-sequencing-p-upf1-rip-seq
#4
Tatsuaki Kurosaki, Mainul Hoque, Lynne E Maquat
Recent progress in the technology of transcriptome-wide high-throughput sequencing has revealed that nonsense-mediated mRNA decay (NMD) targets ~10% of physiologic transcripts for the purpose of tuning gene expression in response to various environmental conditions. Regardless of the eukaryote studied, NMD requires the ATP-dependent RNA helicase upframeshift 1 (UPF1). It was initially thought that cellular NMD targets could be defined by their binding to steady-state UPF1, which is largely hypophosphorylated...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236258/using-tet-off-cells-and-rnai-knockdown-to-assay-mrna-decay
#5
Thomas D Baird, J Robert Hogg
Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236254/integration-of-encode-rnaseq-and-eclip-data-sets
#6
Jorge Boucas
During the last decade, the study of mRNA decay has largely benefited from an increasing number of high-throughput assays that emerged from developments in next generation sequencing (NGS) technologies as well as mass spectrometry. While assay-specific data analysis is often reported and software made available many researchers struggle with the overwhelming challenge of integrating data from diverse assays, different sources, and of different formats.We here use Python, R, and bash to analyze and integrate RNAseq and eCLIP data publicly available from ENCODE...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236253/iclip-of-the-piwi-protein-aubergine-in-drosophila-embryos
#7
Bridlin Barckmann, Jérémy Dufourt, Martine Simonelig
Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs bound to specific Argonaute proteins, the PIWI proteins. piRNAs target mRNAs by complementarity to silence them; they play an important role in the repression of transposable elements in the germ line of many species. piRNAs and PIWI proteins are also involved in diverse biological processes through their role in the regulation of cellular mRNAs. In the Drosophila embryo, they contribute to the maternal mRNA decay occurring during the maternal-to-zygotic transition...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236252/characterizing-mrna-sequence-motifs-in-the-3-utr-using-gfp-reporter-constructs
#8
Rene Geissler, Andrew Grimson
GFP reporter constructs are widely used as an expression system for studying the function of regulatory sequence motifs (cis elements) within the 3'-UTRs (3' untranslated regions) of mRNAs. Here we provide details on the characterization of individual sequence motifs, which typically regulate mRNA decay and translation. In addition, we describe methods to identify trans factors required for the function of such elements. To facilitate efficient identification of novel functional 3'-UTR motifs, we describe a screening approach based on dual-color fluorescence reporter constructs...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236250/measuring-mrna-decay-in-budding-yeast-using-single-molecule-fish
#9
Tatjana Trcek, Samir Rahman, Daniel Zenklusen
Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29236248/determining-mrna-decay-rates-using-rna-approach-to-equilibrium-sequencing-rate-seq
#10
Farah Abdul-Rahman, David Gresham
RATE-seq is a 4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29235495/identification-of-nonsense-mediated-mrna-decay-pathway-as-a-critical-regulator-of-p53-isoform-%C3%AE
#11
Lauren E Cowen, Yi Tang
Human TP53 gene encodes the tumor suppressor p53 and, via alternative splicing, the p53β and γ isoforms. Numerous studies have shown that p53β/γ can modulate p53 functions and are critically involved in regulation of cellular response to stress conditions. However, it is not fully understood how the β and γ isoforms are regulated following splicing. Using gene targeting and RNAi, we showed that depletion of the nonsense-mediated mRNA decay (NMD) factor SMG7 or UPF1 significantly induced p53β but had minimal effect on p53γ...
December 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29225039/a-massively-parallel-reporter-assay-of-3-utr-sequences-identifies-in%C3%A2-vivo-rules-for-mrna-degradation
#12
Michal Rabani, Lindsey Pieper, Guo-Liang Chew, Alexander F Schier
The stability of mRNAs is regulated by signals within their sequences, but a systematic and predictive understanding of the underlying sequence rules remains elusive. Here we introduce UTR-seq, a combination of massively parallel reporter assays and regression models, to survey the dynamics of tens of thousands of 3' UTR sequences during early zebrafish embryogenesis. UTR-seq revealed two temporal degradation programs: a maternally encoded early-onset program and a late-onset program that accelerated degradation after zygotic genome activation...
November 30, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29223154/antiinflammatory-effect-of-rosiglitazone-via-modulation-of-mrna-stability-of-interleukin-10-and-cyclooxygenase-2-in-astrocytes
#13
E V Pankevich, A A Astakhova, D V Chistyakov, M G Sergeeva
Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E2, PGE2) and antiinflammatory marker (interleukin 10, IL-10) levels...
November 2017: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/29222492/interaction-of-rna-binding-protein-hur-and-mir-466i-regulates-gm-csf-expression
#14
Jing Chen, William Adamiak, Ganlei Huang, Ulus Atasoy, Abdolmohamad Rostami, Shiguang Yu
Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3' untranslated region (3'UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α...
December 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29211718/pirna-mediated-regulation-of-transposon-alternative-splicing-in-the-soma-and-germ-line
#15
Felipe Karam Teixeira, Martyna Okuniewska, Colin D Malone, Rémi-Xavier Coux, Donald C Rio, Ruth Lehmann
Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. Here we show that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells...
December 6, 2017: Nature
https://www.readbyqxmd.com/read/29210071/whole-exome-sequencing-of-sickle-cell-disease-patients-with-hyperhemolysis-syndrome-suggests-a-role-for-rare-variation-in-disease-predisposition
#16
Savannah Mwesigwa, Joann M Moulds, Alice Chen, Jonathan Flanagan, Vivien A Sheehan, Alex George, Neil A Hanchard
BACKGROUND: Hyperhemolysis syndrome (HHS) is an uncommon, but life-threatening, transfusion-related complication of red blood cell transfusion. HHS has predominantly been described in patients with sickle cell disease (SCD) and is difficult to diagnose and treat. The pathogenesis of HHS, including its occurrence in only a subset of apparently susceptible individuals, is poorly understood. We undertook whole-exome sequencing (WES) of 12 SCD-HHS patients to identify shared genetic variants that might be relevant to the development of HHS...
December 6, 2017: Transfusion
https://www.readbyqxmd.com/read/29208651/a-12-3-kb-duplication-within-the-vwf-gene-in-pigs-affected-by-von-willebrand-disease-type-3
#17
Stefanie Lehner, Mahnaz Ekhlasi-Hundrieser, Carsten Detering, Hanna Allerkamp, Christiane Pfarrer, Mario von Depka Prondzinski
Von Willebrand Disease (VWD) type 3 is a serious and sometimes fatal hereditary bleeding disorder. In pigs, the disease has been known for decades and affected animals are used as models for the human disease. Due to the recessive mode of inheritance of VWD type 3, severe bleeding is typically seen in homozygous individuals. We sequenced the complete porcine VWF (Von Willebrand Factor) cDNA and detected a tandem duplication of exons 17 and 18, causing a frameshift and a premature termination codon (p.Val814LeufsTer3) in the affected pig...
December 5, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29208426/5-azacytidine-engages-an-ire1%C3%AE-egfr-erk1-2-signaling-pathway-that-stabilizes-the-ldl-receptor-mrna
#18
Nourhen Mnasri, Maya Mamarbachi, Bruce G Allen, Gaétan Mayer
Hepatic low-density lipoprotein receptor (LDLR) is the primary conduit for the clearance of plasma LDL-cholesterol and increasing its expression represents a central goal for treating cardiovascular disease. However, LDLR mRNA is unstable and undergoes rapid turnover mainly due to the three AU-rich elements (ARE) in its proximal 3'-untranslated region (3'-UTR). Herein, our data revealed that 5-azacytidine (5-AzaC), an antimetabolite used in the treatment of myelodysplastic syndrome, stabilizes the LDLR mRNA through a previously unrecognized signaling pathway resulting in a strong increase of its protein level in human hepatocytes in culture...
December 2, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29203639/a-knock-in-tristetraprolin-ttp-zinc-finger-point-mutation-in-mice-comparison-with-complete-ttp-deficiency
#19
Wi S Lai, Deborah J Stumpo, Lianqun Qiu, Roberta Faccio, Perry J Blackshear
Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity, and its ability to promote mRNA decay, are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine to arginine mutation within the first zinc finger...
December 4, 2017: Molecular and Cellular Biology
https://www.readbyqxmd.com/read/29198722/a-recurrent-de-novo-nonsense-variant-in-zswim6-results-in-severe-intellectual-disability-without-frontonasal-or-limb-malformations
#20
Elizabeth E Palmer, Raman Kumar, Christopher T Gordon, Marie Shaw, Laurence Hubert, Renee Carroll, Marlène Rio, Lucinda Murray, Melanie Leffler, Tracy Dudding-Byth, Myriam Oufadem, Seema R Lalani, Andrea M Lewis, Fan Xia, Allison Tam, Richard Webster, Susan Brammah, Francesca Filippini, John Pollard, Judy Spies, Andre E Minoche, Mark J Cowley, Sarah Risen, Nina N Powell-Hamilton, Jessica E Tusi, LaDonna Immken, Honey Nagakura, Christine Bole-Feysot, Patrick Nitschké, Alexandrine Garrigue, Geneviève de Saint Basile, Emma Kivuva, Richard H Scott, Augusto Rendon, Arnold Munnich, William Newman, Bronwyn Kerr, Claude Besmond, Jill A Rosenfeld, Jeanne Amiel, Michael Field, Jozef Gecz
A recurrent de novo missense variant within the C-terminal Sin3-like domain of ZSWIM6 was previously reported to cause acromelic frontonasal dysostosis (AFND), an autosomal-dominant severe frontonasal and limb malformation syndrome, associated with neurocognitive and motor delay, via a proposed gain-of-function effect. We present detailed phenotypic information on seven unrelated individuals with a recurrent de novo nonsense variant (c.2737C>T [p.Arg913Ter]) in the penultimate exon of ZSWIM6 who have severe-profound intellectual disability and additional central and peripheral nervous system symptoms but an absence of frontonasal or limb malformations...
November 23, 2017: American Journal of Human Genetics
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