Abstract:
:Fluorescence live-cell RNA imaging to monitor the intracellular localization and dynamics of the target RNA is a challenging subject. One of the difficulties to achieve this is to establish a precise method to enable a fluorescent labeling to the target RNA in living cells. Technologies to reduce the background fluorescence and to detect the RNA with high sensitivity are also necessary to visualize and analyze the intracellular localization and dynamic of the target RNA precisely. Especially in monitoring single-molecule motion, a special setup of a microscope system is required. Such technical problems make the live-cell RNA imaging to be a difficult subject. We recently developed a methodology to label and to visualize a target RNA in living cells with low background fluorescence by using a probe that is based on an RNA-binding protein domain PUM-HD (pumilio homology domain) and a fluorescent protein reconstitution method. A noteworthy property of PUM-HD to apply RNA probes is that this protein domain can be modified to recognize a particular 8-base RNA sequence by inducing tailor-made designed mutagenesis. The fluorescent protein reconstitution method allows us to detect the target RNA with high signal-to-noise ratio. Using the probe based on PUM-HD, a fluorescent protein reconstitution method, and a homebuilt fluorescent microscope system, we succeeded in single-molecule observation of a target RNA in living cells. In this chapter, the techniques to establish the probe and to observe the motion of single-molecule RNA are described.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Yoshimura H,Ozawa Tdoi
10.1016/bs.mie.2016.03.018subject
Has Abstractpub_date
2016-01-01 00:00:00pages
65-85eissn
0076-6879issn
1557-7988pii
S0076-6879(16)00130-0journal_volume
572pub_type
杂志文章abstract::Monte Carlo simulations of neighbor exclusion models have been used to demonstrate the importance of collecting and fitting data over a wide range of saturation. Low saturation data are important for good estimates of the affinity K of a drug or protein for the lattice site. High saturation data are important for dist...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(94)40065-2
更新日期:1994-01-01 00:00:00
abstract::Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signal...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(04)89005-0
更新日期:2004-01-01 00:00:00
abstract::In recent years, sphingolipid metabolites ceramide, sphingosine, and sphingosine-1-phosphate have emerged as important second messengers in addition to their role as precursors of biomembrane components. The investigation of these sphingolipid metabolites requires the development of new, more sensitive methods for ass...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)34012-3
更新日期:2007-01-01 00:00:00
abstract::Neutron reflectometry (NR) is uniquely positioned in structural biology, because of its ability to characterize biomolecular interfacial architectures like lipid membranes and membrane-associated proteins nondestructively and in their native environment. Mimicking biological processes, samples can be manipulated and t...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.05.019
更新日期:2016-01-01 00:00:00
abstract::Gene therapy vectors based on murine retroviruses have now been in clinical trials for over 20 years. During that time, a variety of novel vector pseudotypes were developed in an effort to improve gene transfer. Lentiviral vectors are now in clinical trials and a similar evolution of vector technology is anticipated. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-386509-0.00005-3
更新日期:2012-01-01 00:00:00
abstract::Drosophila melanogaster is a useful organism for determining protein function and modeling human disease. Drosophila offers a rapid generation time and an abundance of genomic resources and genetic tools. Conservation in protein structure, signaling pathways, and developmental processes make studies performed in Droso...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.028
更新日期:2016-01-01 00:00:00
abstract::We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly t...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.03.013
更新日期:2020-01-01 00:00:00
abstract::Integrin adhesion receptors mediate cell-cell and cell-substratum adhesion and provide a continuous link for the bidirectional transmission of mechanical force and biochemical signals across the plasma membrane. Integrin-dependent cellular activities such as adhesion, migration, proliferation, and survival rely upon t...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)26004-5
更新日期:2007-01-01 00:00:00
abstract::Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data managemen...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)11009-5
更新日期:2006-01-01 00:00:00
abstract::Myoglobin has recently emerged as a versatile metalloprotein scaffold for the design of efficient and selective biocatalysts for abiological carbene transfer reactions, including asymmetric cyclopropanation reactions. Over the past few years, our group has explored several strategies to modulate the carbene transfer r...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.07.007
更新日期:2020-01-01 00:00:00
abstract::Although most eukaryotic mRNAs are degraded by exonucleases acting on either end of the molecule, a subset of mRNAs undergo endonuclease cleavage within the mRNA body. Endonuclease cleavage can be activated by cellular stress, extracellular signals, or by ribosome stalling, as might occur at a premature termination co...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02613-X
更新日期:2008-01-01 00:00:00
abstract::Constitutively activating mutations of the TSHR are the major cause for nonautoimmune hyperthyroidism, which is based on ligand independent, permanent receptor activation. Several reports have highlighted the difficulties to determine whether a TSHR mutation is constitutively active or not especially for borderline ca...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381296-4.00023-3
更新日期:2010-01-01 00:00:00
abstract::Ubiquitination is a prevalent protein modification catalyzed by E1, E2, and E3 enzymes that activate, conjugate, and ligate, respectively, the ubiquitin protein to substrate protein. In order to establish a mutualistic or parasitic relationship with their eukaryotic hosts, many microorganisms hijack different aspects ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.01.007
更新日期:2019-01-01 00:00:00
abstract::Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been stu...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)08012-8
更新日期:2006-01-01 00:00:00
abstract::Autophagy is a catabolic process that is crucial for cellular homeostasis and adaptive response to changing environments. Importantly, autophagy has been shown to be induced in many longevity-associated scenarios and to be required to maintain lifespan extension. Notably, autophagy is a highly conserved cellular proce...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.086
更新日期:2017-01-01 00:00:00
abstract::The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Severa...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391860-4.00013-6
更新日期:2012-01-01 00:00:00
abstract::Increasing numbers of compounds, previously classified as antagonists, were shown to inhibit this spontaneous or constitutive receptor activity, instead of leave it unaffected as expected for a formal antagonist. In addition, some other antagonists did not have any effect by themselves, but prevented the inhibition of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381296-4.00003-8
更新日期:2010-01-01 00:00:00
abstract::In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)09019-1
更新日期:2006-01-01 00:00:00
abstract::The enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are coassembled into higher ordered supercomplexes within the mitochondrial inner membrane. The cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex is formed by the coassociation of the two electron transport chain complexes, the cytochrome ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)04411-X
更新日期:2009-01-01 00:00:00
abstract::Considering that HIV-1 accumulates and replicates actively within lymphoid tissues, any strategy that will decrease viral stores in these tissues might be beneficial to the infected host. Follicular dendritic cells (FDC), B lymphocytes, antigen-presenting cells like macrophages, and activated CD4(+) T cells are abunda...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)91019-7
更新日期:2005-01-01 00:00:00
abstract::When isolating ribonucleoprotein (RNP) complexes by an affinity selection approach, tagging the RNA component can prove to be strategically important. This is especially true for purifying single types of snoRNPs, because in most cases the snoRNA is thought to be the only unique component. Here, we present a general s...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)25014-1
更新日期:2007-01-01 00:00:00
abstract::Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated h...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)06056-3
更新日期:2006-01-01 00:00:00
abstract::For mRNAs encoding secretory and integral membrane proteins, translation initiation is thought to begin a process of mRNA localization where mRNA/ribosome/nascent chain complexes (RNCs) are trafficked from the cytosol compartment to the endoplasmic reticulum (ER). At the ER membrane, RNCs bind to a protein-conducting ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)31004-5
更新日期:2007-01-01 00:00:00
abstract::Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid-protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of thi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)69009-1
更新日期:2009-01-01 00:00:00
abstract::Oligomerization is a frequently encountered physical characteristic of biological molecules that occurs for a wide number of transcription factors, ion channels, oxygen-carrying macromolecules such as hemocyanin and enzymes. On the other hand, unwanted protein oligomerization can lead to the formation of pathogenic st...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)66017-1
更新日期:2009-01-01 00:00:00
abstract::gamma-Glutamyltranspeptidase is a heterodimeric glycoprotein that catalyzes the transpeptidation and hydrolysis of the gamma-glutamyl group of glutathione and related compounds. It is known that the enzyme plays a role in the metabolism of glutathione and in salvaging constituents of glutathione. In the adult animal, ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01025-6
更新日期:2005-01-01 00:00:00
abstract::We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The syst...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/0076-6879(94)35164-3
更新日期:1994-01-01 00:00:00
abstract::The properties of a collection of transposon Tn5 derivatives that generate reporter gene fusions and internal protein tags are summarized. Procedures utilizing several of the transposons for generating genes activated by Cre-loxP recombination and for creating large sequence-defined mutant libraries are described in d...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)21012-7
更新日期:2007-01-01 00:00:00
abstract::Many methods have been developed for the cloning of PCR products. These methods include blunt-end cloning, TA cloning, and using restriction sites incorporated into the PCR primers. The restrictionless cloning technique allows efficient directional cloning of PCR products into any cloning site within a vector regardle...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-418687-3.00009-4
更新日期:2013-01-01 00:00:00
abstract::Growing appreciation for the biogeochemical significance of uncultured microorganisms is changing the focus of environmental microbiology. Techniques designed to investigate microbial metabolism in situ are increasingly popular, from mRNA-targeted fluorescence in situ hybridization (FISH) to the "-omics" revolution, i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381294-0.00012-2
更新日期:2011-01-01 00:00:00