RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins.

Abstract:

:RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Lambert NJ,Robertson AD,Burge CB

doi

10.1016/bs.mie.2015.02.007

subject

Has Abstract

pub_date

2015-01-01 00:00:00

pages

465-493

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(15)00082-8

journal_volume

558

pub_type

杂志文章
  • Mass spectrometry in systems biology an introduction.

    abstract::The qualitative detection, quantification, and structural characterization of analytes in biological systems are important requirements for objectives to be fulfilled in systems biology research. One analytical tool applied to a multitude of systems biology studies is mass spectrometry, particularly for the study of p...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/B978-0-12-385118-5.00002-5

    authors: Dunn WB

    更新日期:2011-01-01 00:00:00

  • Analysis of amyloid aggregates using agarose gel electrophoresis.

    abstract::Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrP(sc) are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)12003-0

    authors: Bagriantsev SN,Kushnirov VV,Liebman SW

    更新日期:2006-01-01 00:00:00

  • Gene expression omnibus: microarray data storage, submission, retrieval, and analysis.

    abstract::The Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information archives and freely distributes high-throughput molecular abundance data, predominantly gene expression data generated by DNA microarray technology. The database has a flexible design that can handle diverse styles of bot...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)11019-8

    authors: Barrett T,Edgar R

    更新日期:2006-01-01 00:00:00

  • Stem cells in the lung.

    abstract::The lung is composed of two major anatomically distinct regions-the conducting airways and gas-exchanging airspaces. From a cell biology standpoint, the conducting airways can be further divided into two major compartments, the tracheobronchial and bronchiolar airways, while the alveolar regions of the lung make up th...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)19012-6

    authors: Liu X,Driskell RR,Engelhardt JF

    更新日期:2006-01-01 00:00:00

  • Strategies to rationalize enzyme immobilization procedures.

    abstract::Enzyme immobilization is a widespread empiric technology to achieve more stable, active and reusable enzymes. The empiricism can be reduced by the application of rational design procedures employing bioinformatic tools, engineered-proteins and detailed analyses of existent data. In this chapter, we describe relevant a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.09.003

    authors: Sastre DE,Reis EA,Marques Netto CGC

    更新日期:2020-01-01 00:00:00

  • Molecular tools for investigating ANME community structure and function.

    abstract::Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabol...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385112-3.00004-4

    authors: Hallam SJ,Pagé AP,Constan L,Song YC,Norbeck AD,Brewer H,Pasa-Tolic L

    更新日期:2011-01-01 00:00:00

  • Use of sec mutants to define intermediates in protein transport from endoplasmic reticulum.

    abstract::In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion. The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, additi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(92)19028-5

    authors: Rexach MF,Schekman RW

    更新日期:1992-01-01 00:00:00

  • VEGF receptor signal transduction.

    abstract::Signal transduction by vascular endothelial growth factors (VEGFs) through their cognate VEGF receptor tyrosine kinases follows the consensus scheme for receptor tyrosine kinases. Thus, binding of ligand induces receptor dimerization and activation of the tyrosine kinase through transphosphorylation between receptor m...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)02013-2

    authors: Li X,Claesson-Welsh L,Shibuya M

    更新日期:2008-01-01 00:00:00

  • Manipulation of PKC isozymes by RNA interference and inducible expression of PKC constructs.

    abstract::Protein kinase C (PKC), a family of serine/threonine kinases, plays an important role in apoptosis. Several members of the PKC family act as substrates for caspases. In addition, PKCs can also regulate caspase activation and cell death by apoptosis. The cleavage of PKCs separates the regulatory domain from the catalyt...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)01608-X

    authors: Basu A,Persaud SD,Sivaprasad U

    更新日期:2008-01-01 00:00:00

  • The use of in vitro assays to measure endoplasmic reticulum-associated degradation.

    abstract::Approximately one-third of all newly translated polypeptides interact with the endoplasmic reticulum (ER), an event that is essential to target these nascent proteins to distinct compartments within the cell or to the extracellular milieu. Thus, the ER houses molecular chaperones that augment the folding of this diver...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(10)70027-6

    authors: Brodsky JL

    更新日期:2010-01-01 00:00:00

  • Predicting fluorescence lifetimes and spectra of biopolymers.

    abstract::Use of fluorescence in biology and biochemistry for imaging and characterizing equilibrium and dynamic processes is growing exponentially. Much progress has been made in the last few years on the microscopic understanding of the underlying principles of what controls the wavelength and quenching of fluorescence in bio...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-381270-4.00001-9

    authors: Callis PR

    更新日期:2011-01-01 00:00:00

  • Enzymatic synthesis of functional amylosic materials and amylose analog polysaccharides.

    abstract::Because polysaccharides have very complicated chemical structures constructed by a great diversity of monosaccharide residues and glycosidic linkages, enzymatic approaches have been identified as powerful tools to precisely synthesize polysaccharides as the reactions progress in highly controlled regio- and stereoarra...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.06.009

    authors: Kadokawa JI

    更新日期:2019-01-01 00:00:00

  • Reversible Chemical Dimerization by rCD1.

    abstract::An optimal tool to unravel the role of a specific player within a cellular network or process requires its spatiotemporally resolved perturbation. Chemically induced dimerization (CID) by the rapamycin system has proven useful to induce protein dimerization or translocation with high spatiotemporal precision. Recently...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.10.035

    authors: Schifferer M,Feng S,Stein F,Schultz C

    更新日期:2017-01-01 00:00:00

  • RNA interference techniques to study epithelial cell adhesion and polarity.

    abstract::Polarized epithelial cells are characterized by distinct plasma membrane domains and asymmetrical distribution of cell surface proteins and lipids. In vertebrates, tight junctions act as a fence between the apical and basolateral domains. Although many of the key components of the polarity machinery have been identifi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)06026-5

    authors: Chen X,Macara IG

    更新日期:2006-01-01 00:00:00

  • Reactions of nitric oxide and oxygen with the regulator of fumarate and nitrate reduction, a global transcriptional regulator, during anaerobic growth of Escherichia coli.

    abstract::The Escherichia coli fumarate and nitrate reductase (FNR) regulator protein is an important transcriptional regulator that controls the expression of a large regulon of more than 100 genes in response to changes in oxygen availability. FNR is active when it acquires a [4Fe-4S](2+) cluster under anaerobic conditions. T...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)37011-0

    authors: Crack JC,Le Brun NE,Thomson AJ,Green J,Jervis AJ

    更新日期:2008-01-01 00:00:00

  • Gene knockouts, in vivo site-directed mutagenesis and other modifications using the delitto perfetto system in Saccharomyces cerevisiae.

    abstract::Gene manipulation serves the purpose of providing a better understanding of the function of specific genes as well as for developing novel variants of the genes of interest. The generation of knockout genes, the alteration, depletion, or enhancement of a particular gene function through the generation of specific gene...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-420067-8.00008-8

    authors: Stuckey S,Storici F

    更新日期:2013-01-01 00:00:00

  • Continuum Electrostatics Approaches to Calculating pKas and Ems in Proteins.

    abstract::Proteins change their charge state through protonation and redox reactions as well as through binding charged ligands. The free energy of these reactions is dominated by solvation and electrostatic energies and modulated by protein conformational relaxation in response to the ionization state changes. Although computa...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/bs.mie.2016.05.052

    authors: Gunner MR,Baker NA

    更新日期:2016-01-01 00:00:00

  • Measurement of mitochondrial turnover and life cycle using MitoTimer.

    abstract::Current methodologies available to quantify changes in mitochondrial turnover are limited to pulse-chase assays or specific assays that quantify mitophagy. Accordingly, new tools that can assess mitochondrial turnover are needed for the study of cellular, subcellular, and spatial parameters of mitochondrial turnover a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-801415-8.00002-3

    authors: Trudeau KM,Gottlieb RA,Shirihai OS

    更新日期:2014-01-01 00:00:00

  • Functional Characterization of Histone Chaperones Using SNAP-Tag-Based Imaging to Assess De Novo Histone Deposition.

    abstract::Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specifici...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.04.004

    authors: Clément C,Vassias I,Ray-Gallet D,Almouzni G

    更新日期:2016-01-01 00:00:00

  • Construction of immunotoxins using Pseudomonas exotoxin A.

    abstract::The above methodology has been used to prepare a variety of PE-immunotoxins. These reagents are potent cytotoxic agents for cells in culture that bind the appropriate antibody and nontoxic when cells do not bind the antibody. PE-immunotoxins can be used to select for mutant cultured cells which lack the target of the ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(87)51015-1

    authors: FitzGerald DJ

    更新日期:1987-01-01 00:00:00

  • In Vitro Analysis of Cyanobacterial Nonheme Iron-Dependent Aliphatic Halogenases WelO5 and AmbO5.

    abstract::Aliphatic carbon-halogen (C-X) bonds are prevalent in modern pharmaceuticals and bioactive natural products. Three distinct chemical strategies are known in Nature to generate these structural motifs. The first is via the nucleophilic substitution at a prefunctionalized electrophilic carbon center with a halide anion ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.02.015

    authors: Liu X

    更新日期:2018-01-01 00:00:00

  • Studying trans-acting enzymes that target carrier protein-bound amino acids during nonribosomal peptide synthesis.

    abstract::Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer p...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.12.008

    authors: Greule A,Charkoudian LK,Cryle MJ

    更新日期:2019-01-01 00:00:00

  • Detection and characterization of peroxynitrite-induced modifications of tyrosine, tryptophan, and methionine residues by tandem mass spectrometry.

    abstract::Nitration and oxidation of tyrosine, tryptophan, and methionine residues in proteins are potential markers of their interaction with peroxynitrite. This chapter describes the procedure for the detection of these nitro-oxidative modifications by tandem mass spectrometry. The peptide YGDLANWMIPGK, shown to contain a nit...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(08)01215-9

    authors: Rebrin I,Bregere C,Gallaher TK,Sohal RS

    更新日期:2008-01-01 00:00:00

  • Liposomalized oligopeptides in cancer therapy.

    abstract::Organ-specific delivery of biofunctional agents is thought to enhance their activity and to reduce their side effects. Liposomes have been used as drug carriers in cancer chemotherapy, since they accumulate passively in tumor tissues due to an enhanced permeability and retention (EPR) effect. In addition, modification...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)91009-4

    authors: Asai T,Oku N

    更新日期:2005-01-01 00:00:00

  • Cellular Assays for Evaluating Calcium-Dependent Translocation of cPLA2α to Membrane.

    abstract::The group IVA phospholipase A2, commonly called cytosolic phospholipase A2α (cPLA2α), is a widely expressed enzyme that hydrolyzes membrane phospholipid to produce arachidonic acid and lysophospholipids, which are precursors for a number of bioactive lipid mediators. Arachidonic acid is metabolized through the cycloox...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.09.006

    authors: Yun B,Leslie CC

    更新日期:2017-01-01 00:00:00

  • Analysis of autophagy during infections of Cryptococcus neoformans.

    abstract::Cryptococcus neoformans is a yeastlike fungus that causes a lethal meningoencephalitis in a broad spectrum of immunocompromised patients and has become the most common cause of meningitis due to AIDS-related infections in Africa. Key to the development of new agents to control and prevent this infection is the identif...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)03222-9

    authors: Hu G,Gibbons J,Williamson PR

    更新日期:2008-01-01 00:00:00

  • Phylogenetic analysis and experimental approaches to study color vision in vertebrates.

    abstract::To elucidate the molecular mechanisms of vertebrate color vision, it is essential to establish associations between amino acid substitutions and the directions of lambda max shifts of visual pigments. In this way, we can identify critical amino acid changes that may be responsible for lambda max shifts of visual pigme...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(00)15851-3

    authors: Yokoyama S

    更新日期:2000-01-01 00:00:00

  • Retinoic acid and microRNA.

    abstract::Retinoic acid (RA), the biologically active metabolite of vitamin A, regulates a vast spectrum of biological processes, such as cell differentiation, proliferation, apoptosis, and morphogenesis. microRNAs (miRNAs) play a crucial role in regulating gene expression by binding to messenger RNA (mRNA) which leads to mRNA ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.02.009

    authors: Wang L,Rohatgi AP,Wan YY

    更新日期:2020-01-01 00:00:00

  • Selective amino acid segmental labeling of multi-domain proteins.

    abstract::The steady technical advances of nuclear magnetic resonance (NMR) over the past decades enabled a significant increase in the molecular size of protein particles that can be subjected to a structural and functional characterization in solution. The larger molecular weight of such proteins is accompanied with an increa...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.05.028

    authors: Michel E,Allain FH

    更新日期:2015-01-01 00:00:00

  • Preparation and differentiation of mesenchymal stem cells from ears of adult mice.

    abstract::External murine ears collected postmortem, as well as ear punches obtained during standard marking of live animals, are the source of mesenchymal stem cells, termed ear mesenchymal stem cells (EMSC). These cells provide an easily obtainable, primary culture model system for the study of lineage commitment and differen...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-800280-3.00001-3

    authors: Gawronska-Kozak B

    更新日期:2014-01-01 00:00:00