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Ubiquitin stress-induced NEDDylation leads to the formation of aggresome-like bodies (ALBs) in the perinuclear region of cells. Therefore, imaging analysis is essential for characterizing the biological phenotypes of ALBs. Here, we describe a protocol to monitor ALBs induced by ubiquitin stress using immunocytochemistry and to quantify cells containing ALBs. This optimized protocol details the use of readily available materials and reagents and can be applied to explore diverse molecules involved in stress-induced ALBs. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The cyclic GMP-AMP synthase (cGAS) is the principal DNA sensor, which binds DNA and triggers the type I interferon production. We used ISD45 or inactivated Vaccinia Virus (VACV) to stimulate cGAS and monitored cellular localization by immunofluorescence microscopy, Operetta high-content screening, and cytoplasmic/nuclear fractionation. LocNES server was used to predict cGAS nuclear export signal (NES) sequence and characterized the function by mutagenesis. This protocol provides a prototype of cGAS subcellular distribution or the identification of NES in other proteins. For complete details on the use and execution of this protocol, please refer to Sun et al. Sun et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5β1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.

RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein-RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically enhance coverage and to reach single amino acid resolution. Moreover, the simple mono-nucleoside adducts offer a confident and quantitative measure of direct RNA-protein interaction. Using RBS-ID, we profiled ~2,000 human RBSs and probed Streptococcus pyogenes Cas9 to discover residues important for genome editing.

For enteroviruses such as poliovirus (PV), empty capsids, which are antigenically indistinguishable from mature virions, are produced naturally during viral infection. The production of such capsids recombinantly, in heterologous systems such as yeast, have great potential as virus-like particle (VLP) vaccine candidates. Here, using PV as an exemplar, we show the production of VLPs in Pichia pastoris by coexpression of the structural precursor protein P1 and the viral protease 3CD. The level of expression of the potentially cytotoxic protease relative to that of the P1 precursor was modulated by three different approaches: expression of the P1 precursor and protease from different transcription units, separation of the P1 and protease proteins using the Thosea asigna virus (TaV) 2A translation interruption sequence, or separation of the P1 and protease-coding sequences by an internal ribosome entry site sequence from Rhopalosiphum padi virus (RhPV). We also investigate the antigenicity of VLPs containing previously characterized mutations when produced in Pichia Finally, using transmission electron microscopy and two-dimensional classification, we show that Pichia -derived VLPs exhibited the classical icosahedral capsid structure displayed by enteroviruses. IMPORTANCE Although the current poliovirus immunization program has been extremely successful in reducing the number of cases of paralytic polio worldwide, now more cases are caused by vaccine-derived polioviruses than by wild poliovirus. Switching to inactivated poliovirus vaccines will reduce this over time; however, their production requires the growth of large amounts of virus. This biosafety concern can be addressed by producing just the virus capsid. The capsid serves to protect the genetic material, which causes disease when introduced into a cell. Therefore, empty capsids (virus-like particles [VLPs]), which lack the viral RNA genome, are safe both to make and to use. We exploit yeast as a versatile model expression system to produce VLPs, and here we specifically highlight the potential of this system to supply next-generation poliovirus vaccines to secure a polio-free world for the future., (Copyright © 2020 Sherry et al.)

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 have recently emerged as important players in regulating innate and adaptive immune responses by trimming peptide ligands for MHC class I molecules. Functional polymorphisms in ERAP1 and ERAP2 genes have been associated with predisposition to several diseases including autoimmune diseases, viral infections, and virally induced cancers. In this chapter, we describe two basic methods for monitoring peptide-trimming activity by ER aminopeptidases and screening potential chemical inhibitors.

β-Arrestins (β-arrs) were originally appreciated for the roles they play in the desensitization and internalization of G protein-coupled receptors (GPCRs). They are also now known to act as molecular scaffolds, providing control in multiple signalling pathways. Through their scaffolding properties, β-arrs dynamically regulate the activity and/or subcellular distribution of protein partners giving rise to an appropriate cellular response. There are two β-arr isoforms, namely, β-arr1 and β-arr2, which share high sequence homology and structural conservation. While the β-arrs often display conserved overlapping roles, decisive differences between the isoforms also exist. A striking example of this is the subcellular distribution of the β-arr isoforms. While β-arr1 is distributed both in cytoplasmic and nuclear compartments, β-arr2 displays an apparent cytoplasmic distribution. Both β-arrs are actively imported into the nucleus, but β-arr2 is constitutively exported by a leptomycin B-sensitive pathway due to a nuclear export signal in its C-terminus that is absent in β-arr1. β-arr2 therefore undergoes constitutive nucleocytoplasmic shuttling enabling the displacement of nuclear binding cargoes, such as Mdm2. Here, we describe methods to explore the differential nucleocytoplasmic shuttling capacities of the β-arrs.

Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to confer greater biostability. We have recently developed a new MB architecture composed of 2'-O-methyl RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester stem (2Me/PS LOOP MB). We showed that this new MB exhibits a marginal level of false-positive signals and enables accurate single-molecule imaging of target RNA in living cells. In this chapter, we describe detailed methods that led us to conclude that, among various PS-modified configurations, the 2Me/PS LOOP MB is an optimal design for intracellular RNA analysis.

Heat shock proteins (HSP) are rapidly induced after stresses such as heat shock and accumulate at high concentrations in cells. HSP induction involves primarily a family of heat shock transcription factors (HSF) that bind the heat shock elements of the HSP genes and mediate transcription in trans. We discuss methods for the study of HSP binding to HSP promoters and the consequent increases in HSP gene expression in vitro and in vivo.

Distinct histone variants mark chromatin domains in the nucleus. To understand how these marks are established and maintained, one has to decipher how the dynamic distribution of these variants is orchestrated. These dynamics are associated with all DNA-based processes such as DNA replication, repair, transcription, heterochromatin formation and chromosome segregation. Key factors, known as histone chaperones, have been involved in escorting histones, thereby contributing to the chromatin landscape of given cell types. SNAP-tag-based imaging system enables the distinction between old and newly deposited histones, and has proved to be a powerful method for the visualization of histone variant dynamics on a cell-by-cell basis. This approach enables the tracking of specific variants in vivo and defining their timing and mode of deposition throughout the cell cycle and in different nuclear territories. Here, we provide a detailed protocol to exploit the SNAP-tag technology to assess the dynamics of newly synthesized and old histones. We then show that combining the SNAP-tagging of histones with the knockdown of candidate factors, represents an effective approach to decipher the role of key actors in guiding histone dynamics. Here, we specifically illustrate how this strategy was used to identify the essential role of the chaperone HIRA in deposition of newly synthesized histone variant H3.3.

Human HspB1 (also denoted as Hsp27) belongs to the family of small (or stress) proteins (sHsps). The family, which contains ten members including αA,B-crystallin polypeptides, is characterized by a conserved C-terminal α-crystallin domain and molecular weights ranging from 20 to 40 kDa. Here, procedures are described for analyzing the dynamic oligomerization and phosphorylation patterns of HspB1 in cells exposed to different environments. Changes in the structural organization of HspB1 can reprogram its interaction with specific partner/client polypeptides. Methods are presented to analyze these interactions using tissue culture cells genetically modified to express different levels of this protein. In addition, the laboratory approaches presented here could be used to test the nine other human sHsp members as well as sHsps from other species.

Identification of the interaction partners of a protein is one of useful straightforward methods to gain insight into the molecular functions of the protein in cells. The pre-deposited forms of histones are associated with the specific histone chaperones to assemble into chromatin. Here, I describe an affinity purification method using the FLAG/HA double epitope-tagging technique and its application to purify particular histone variant-interacting chaperone complexes from soluble fraction to study dynamic chromatin functions. The purification is performed under low salt condition to obtain native histone variant complexes, and it would be useful to identify the specific chaperone proteins involved in the specific chromatin functions via histone variants.

The study of histone variants and histone posttranslational modifications (PTMs) is a trending topic in different fields including developmental biology, neurobiology, and immunology; as well as in the understanding of molecular mechanisms leading to diverse diseases, such as cancer. Since the establishment of histone PTMs starts immediately after their synthesis and it continues once they are assembled into chromatin, here we describe a classic protocol of subcellular fractionation aiming to study histones at different stages of maturation. This includes newly synthesized histones enriched in cytosolic fractions; a pool of newly synthesized, evicted, and stored histones enriched in nuclear soluble fractions; and chromatin-associated histones enriched in chromatin pellet. To study specific histone variants and the establishment of their PTMs, we describe a protocol for obtaining histone variants expressed in bacteria. In addition, we describe a Triton-Acetic acid-Urea (TAU) gel electrophoresis protocol adapted to work on mini-gels, which can be coupled to Western blot to analyze PTMs on histone variants. Finally, we describe a Chromatin immunoprecipitation (ChIP) assay for studying histone PTMs, or tagged histone variants, on specific DNA sequences.

Mitochondria dysfunction plays a role in many human diseases. Therapeutic techniques for these disorders require novel delivery systems that can specifically target and penetrate mitochondria. In this study, we report a novel nanosome composed of dequalinium-DOTAP-DOPE (1,2 dioleoyl-3-trimethylammonium-propane-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (DQA80s) as a potential mitochondria-targeting delivery vector. The functional DQAsome, DQA80s, showed enhanced transfection efficiency compared to a vector DQAsomes in HeLa cells and dermal fibroblasts. In addition, DQA80s/pDNA complexes exhibited rapid escape from the endosome into the cytosol. We observed the delivery of pDNA to mitochondria in living cells using flow cytometry, confocal microscopy, and TME imaging. More specifically, we confirmed our results by co-localization of hmtZsGreen constructs to mitochondria when delivered via DQAsomes and DQA80s in living cells. The mitochondria-targeting DQAsomes and DQA80s induced mitochondrial dysfunction through depolarization of mitochondrial membrane potential. Our data demonstrate that DQA80s show promise for use as a mitochondria-targeted carrier system for treatment of mitochondria diseases in vivo., (Copyright © 2017. Published by Elsevier B.V.)

The last step of cytokinesis, abscission, consists in the severing of the intercellular bridge connecting the two daughter cells. Because daughter cells move randomly on regular cell culture substrates, the use of adhesive micropatterns facilitates the observation of the intercellular bridge and its severing. Here we propose general rules to design micropatterns optimized to study this process. In particular, these micropatterns allow a good stabilization of the daughter cells and a predictable positioning of the intercellular bridge. We suggest a series of micropatterns controlling various cellular parameters such as distance between daughter cells or daughter cells polarization. We give recommendations for videomicroscopy acquisition during cell division and propose automated image analysis methods using kymograph analysis or bridge detection. Finally, we detail methods to artificially cut the intercellular bridge using UV-based laser ablation or using two-photons laser ablation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term "controlled unmasking of targeting elements" (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin-biotin system. The targeting signal is generated within or immediately after a 38-amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum., (© 2016 Abraham, Gotliv, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The role of cyclin-dependent kinase inhibitors (CDKIs) is to negatively regulate cyclin-dependent kinases as a mechanism of control of cell proliferation. As such, CDKIs are being used to induce apoptosis in cancer cells to prevent their excessive reproduction. This chapter describes procedures to study apoptosis induction upon treatment with any CDKI through the evaluation of morphological and functional mitochondrial alterations, in particular, how to measure the mitochondrial membrane potential (ΔΨm) using TMRE dye, determine the content of intracellular ATP, observe mitochondrial network morphology using HeLa cells stably expressing fluorescent reporter DsRed targeting mitochondrial matrix, observe ultrastructure of the organelle using transmission electron microscopy, and, finally, assure that mitochondrial outer membrane permeabilization takes place by assessing the subcellular localization of cyt C in HeLa cells stably expressing fluorescent cyt C-GFP.

RNA associates with RNA-binding proteins (RBPs) from synthesis to decay, forming dynamic ribonucleoproteins (RNPs). In spite of the preeminent role of RBPs regulating RNA fate, the scope of cellular RBPs has remained largely unknown. We have recently developed a novel and comprehensive method to identify the repertoire of active RBPs of cultured cells, called RNA interactome capture. Using in vivo UV cross-linking on cultured cells, proteins are covalently bound to RNA if the contact between the two is direct ("zero distance"). Protein-RNA complexes are purified by poly(A) tail-dependent oligo(dT) capture and analyzed by quantitative mass spectrometry. Because UV irradiation is applied to living cells and purification is performed using highly stringent washes, RNA interactome capture identifies physiologic and direct protein-RNA interactions. Applied to HeLa cells, this protocol revealed the near-complete repertoire of RBPs, including hundreds of novel RNA binders. Apart from its RBP discovery capacity, quantitative and comparative RNA interactome capture can also be used to study the responses of the RBP repertoire to different physiological cues and processes, including metabolic stress, differentiation, development, or the response to drugs.

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Autophagy is an important catabolic pathway that preserves cellular homeostasis. The formation of autophagosomes is a complex process requiring the reorganization of membranes from different compartments. Here we describe methods to analyze SNARE-dependent vesicular fusion events involving the homotypic and heterotypic fusion of autophagosome precursor structures. These two steps are essential for the maturation of small single-membrane autophagic precursors containing ATG16L1 and mATG9 proteins into double-membrane autophagosomes. The techniques described in this review are mostly based on live cell imaging, microscopy, and biochemistry using an in vitro fusion assay, and should help researchers to study autophagosome biogenesis., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

A wide variety of noncoding RNAs are thought to play fundamental and regulatory functions in the mammalian cell nucleus, although their primary functions have yet to be elucidated. Here, we describe a method for the rapid and efficient elimination of specific nuclear noncoding RNAs in mammalian cells using chemically modified antisense oligonucleotides, which may facilitate the functional assessment of nuclear ncRNAs.

Studies over the past several years have revealed that steps in gene expression are extensively coupled to one another both physically and functionally. Recently, in vitro systems were developed for understanding the mechanisms involved in coupling transcription by RNA polymerase II to RNA processing. Here we describe an efficient two-way system for coupling transcription to splicing and a robust three-way system for coupling transcription, splicing, and polyadenylation. In these systems a CMV-DNA construct is incubated in HeLa cell nuclear extracts in the presence of (32)P-UTP to generate the nascent transcript. Transcription is then stopped by addition of α-amanitin followed by continued incubation to allow RNA processing.

Kinetic analysis of in vitro splicing is a valuable technique for understanding splicing regulation. It allows the determination of specific contributions from functional elements for the efficient removal of introns. This chapter will describe the rationale and approach employed to use kinetic analysis to evaluate an in vitro splicing reaction using radiolabeled pre-mRNA incubated in splicing-competent HeLa nuclear extract (NE).

The in vitro splicing assay is a valuable technique that can be used to study the mechanism and machinery involved in the splicing process. The ability to investigate various aspects of splicing and alternative splicing appears to be endless due to the flexibility of this assay. Here, we describe the tools and techniques necessary to carry out an in vitro splicing assay. Through the use of radiolabeled pre-mRNA and crude nuclear extract, spliced mRNAs can be purified and visualized by autoradiography for downstream analysis.

Isolating spliceosomes at a specific assembly stage requires a means to stall or enrich for one of the intermediate splicing complexes. We describe strategies to arrest spliceosomes at different points of complex formation and provide a detailed protocol developed for isolating intact splicing complexes arrested between the first and second chemical steps of splicing. Briefly, spliceosomes are assembled on a radiolabeled in vitro-transcribed splicing substrate from components present in nuclear extract of HeLa cells. Spliceosome progression is arrested after the first step of splicing chemistry by mutating the pre-mRNA substrate at the 3' splice site. The substrate also contains binding sites for the MS2 protein, which serve as an affinity tag. Purification of arrested spliceosomes is carried out in two steps: (1) size exclusion chromatography and (2) affinity selection via a fusion of MS2 and maltose-binding protein (MBP). Complex assembly and purification are analyzed by denaturing polyacrylamide gel electrophoresis.

Splicing components play an essential role in mediating accurate and efficient splicing. The complexity of the spliceosome and its regulatory networks increase the difficulty of studying the splicing reaction in detail. Nuclear extracts derived from HeLa cells provide all of the obligatory components to carry out intron removal in vitro. This chapter describes the large-scale preparation of nuclear extract from HeLa cells.

Pre-mRNA splicing is a key step for generating mature protein-coding mRNA. An RNA-protein complex known as the spliceosome carries out the chemistry of pre-mRNA splicing. However, several pre-spliceosomal intermediates are assembled on the pre-mRNA before the formation of the catalytically activated spliceosome. The progression to the activated spliceosome involves a cascade of the rearrangement events of the RNA-RNA, RNA-protein, and protein-protein interactions within the pre-spliceosomal intermediates. These rearrangements generate multiple combinatorial interactions of the spliceosome with the substrate, which enhances the accuracy of the splice site selection. Each rearrangement also represents a step at which splicing can potentially be subjected to regulation. The aim of this chapter is to provide an overview of the components of the spliceosome and their rearrangements along the spliceosome assembly pathway.

The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

Epigenetic inheritance is the inheritance of genetic information that is not based on DNA sequence alone. One type of epigenetic information that has come to the forefront in the last few years is modified DNA bases. The most common modified DNA base in nature is 5-methylcytosine. Herein, we describe a laboratory experiment that combines bioinformatic and molecular approaches to study the presence and abundance of 5-methylcytosine in different organisms. Students were originally provided with the protein sequence of the Xenopus laevis DNMT1 cytosine-5 DNA methyltransferase and used BLASTP searches to detect the presence of protein orthologs in the genomes of several organisms including Homo sapiens, Mus musculus, Plasmodium falciparum, Drosophila melanogaster, Saccharomyces cerevisiae, Arabidopsis thaliana, and Caenorhabditis elegans. Students generated hypotheses regarding the presence and abundance of 5-methylcytosine in these organisms based on their bioinformatics data, and directly tested their predictions on a subset of DNAs using restriction enzyme isoschizomer assays. A southern blotting assay to answer the same question is also presented. In addition to exposure to the field of epigenetics, the strengths of the laboratory are students are able to make predictions using bioinformatic tools and quickly test them in the laboratory. In addition, students are exposed to two potential misinterpretations of bioinformatic search data. The laboratory is easily modified to incorporate outside research interests in epigenetics., (© 2013 by The International Union of Biochemistry and Molecular Biology.)

Human autoantibodies were a key to the discovery of GW bodies and their integral protein, GW182. This publication marks the tenth anniversary of the discovery of GW182. As it turns out, the discovery of GW182 was quite timely because it coincided with the elucidation of the RNA interference (RNAi) pathway, which is now known to have a major role in post-transcriptional gene regulation. Following our publication of the essential features of GW182 in 2002, laboratories from around the world began investigations that led to the elucidation of the role of GW182 in RNAi and other pathways of mRNA processing and degradation. This chapter reviews the discovery of GW182 and the description of GWB and some of the observations that followed that still remain to be elucidated.

Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) enables the quantitative study of cell adhesion under physiological conditions. SCFS probes adhesive interactions of single living cells with substrates such as extracellular matrix (ECM) proteins and other cells. Here, we present a protocol to quantitatively study the adhesion of HeLa cells to covalently immobilized fibronectin and Matrigel™ using SCFS. We describe procedures for (a) functionalization of AFM cantilevers, (b) preparation of maleic anhydride copolymer thin films, (c) covalent immobilization of ECM proteins on the thin films, (d) cell handling and attachment to the AFM cantilever, and (e) measurement of adhesion forces. The protocol can be easily modified for other cell types and substrate proteins.

Positioning of nucleosomes regulates the access of DNA binding factors to their consensus sequences. Nucleosome positions are determined, at least in part by the effects of DNA sequence during nucleosome assembly. Nucleosomes can also be repositioned (moved in cis) by ATP-dependent nucleosome remodeling complexes. Most studies of repositioning have used short DNA fragments containing a single nucleosome. It is difficult to use this type of template to analyze the role of DNA sequence in repositioning, however, because the many remodeling complexes are strongly influenced by nearby DNA ends. Mononucleosomal templates also cannot provide information about how repositioning occurs in the context of chromatin, where the presence of flanking nucleosomes could limit repositioning options. This protocol describes a newly developed method that allows the mapping of nucleosome positions (with and without remodeling) on any chosen region of a plasmid polynucleosomal template in vitro. The approach uses MNase digestion to release nucleosome-protected DNA fragments, followed by restriction enzyme digestion to locally unique sites, and Southern blotting, to provide a comprehensive map of nucleosome positions within a probe region. It was developed as part of studies which showed that human remodeling enzymes tended to move nucleosomes away from high affinity nucleosome positioning sequences, and also that there were differences in repositioning specificity between different remodeling complexes.

The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.

An eGFP-intron splicing system that allows for co-ordinated expression of up to four siRNAs from a single adenoviral vector has been developed. In this splicing structure the intron, embedded by a multiple miR30-based shRNAs, is located between two incomplete eGFP domains which require successful splicing for functionality. To prove the principle of the method, an adenoviral vector delivering four transcripts targeting survivin, XIAP, Hec1, and VEGF was developed which enabled the knockdown of target genes by 70, 70, 54 and 44%, respectively, in HeLa cells. This is the first report of multi-siRNA engineering technology in the context of adenoviral vector which would enable concomitant knockdown of tumor-related target genes. The results provide a strategy for gene function analysis and cancer gene therapy., (© Springer Science+Business Media B.V. 2011)

Background: The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems., Methods: Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s)., Results: It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s), which corrected the splicing defects., Conclusions: Splice-switch technology, originally developed for genetic disease therapy, can also be used to control gene expression of viral vectors. This approach represents a novel, universal and powerful method for controlling gene expression, replication, viral spread and, by extension, virus-induced cytotoxic effects and can be used both for basic studies of virus infection and in virus-based gene- and anti-cancer therapy.

Since gene expression is controlled on many different levels in a cell, capturing a comprehensive snapshot of all regulatory processes is a difficult task. One possibility to monitor effective changes within a cell is to directly quantify changes in protein synthesis, which reflects the accumulative impact of regulatory mechanisms on gene expression. Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) has been shown to be a viable method to investigate de novo protein synthesis on a proteome-wide scale (Schwanhausser et al., Proteomics 9:205-209, 2009; Selbach et al., Nature 455:58-63, 2008). One application of pSILAC is to study the regulation of protein expression by microRNAs. Here, we describe how pSILAC in conjunction with shotgun mass spectrometry can assess differences in the protein profile between cells transfected with a microRNA and non-transfected cells.

The G-less cassette is a 365-nucleotide (nt) segment of DNA lacking guanine (G) residues on the nontemplate strand. In principle, a full-length transcript can be generated in an in vitro reaction lacking GTP, an omission that leads to suppression of most random, nonspecific transcription throughout the plasmid. This method, like runoff transcription, generates radiolabeled RNA products, directly bypassing the necessity and extra time required to perform primer extension or other indirect mRNA product measurements. Unlike runoff transcription, which requires a cleaved end, the G-less assay can be performed on a circular, supercoiled plasmid, which in many systems is a more efficient template. In practice, most crude systems, such as HeLa nuclear extracts, contain low amounts of contaminating GTP that lead to small amounts of background transcription and occasionally can cause random upstream transcription to read through the G-less cassette. To minimize these artifacts, the reaction generally contains 3'-O-Me-GTP, a chain-terminating analog of GTP that causes transcription to cease when it is incorporated into a growing transcript, much like the dideoxy analogs used in DNA sequencing. The reaction products are cleaved with T1 RNase, which cleaves RNAs at G residues, further reducing background transcription; the G-less mRNA remains intact, whereas small random RNAs are digested.

In the past 10 years, transcriptome and proteome analyses have provided valuable data on global gene expression and cell functional networks. However, when integrated,these analyses revealed partial correlations between mRNA expression levels and protein abundance thus suggesting that post-transcriptional regulations may be in part responsible for this discrepancy. In the present work, we report the development of a functional, integrated, and quantitative method to measure post-transcriptional regulations that we named FunREG. This method enables (i) quantitative measure of post-transcriptional regulations mediated by selected 3-untranslated regions and exogenous small interfering-RNA or micro-RNAs and (ii) comparison of these regulatory processes in physiologically relevant systems (e.g. cancer versus primary untransformed cells). We applied FunREG to the study of liver cancer, and we demonstrate for the first time the differential regulatory mechanisms controlling gene expression at a post-transcriptional level in normal and tumoral hepatic cells. As an example, translation efficiency mediated by heparin-binding epidermal growth factor 3-untranslated region was increased 3-fold in liver cancer cells compared with normal hepatocytes, whereas stability of an mRNA containing a portion of Cyclin D1 3-untranslated region was increased more than 2-fold in HepG2 cells compared with normal hepatocytes. Consequently we believe that the method presented herein may become an important tool in fundamental and medical research. This approach is convenient and easy to perform, accessible to any investigator, and should be adaptable to a large number of cell type, functional and chemical screens, as well as genome scale analyses. Finally FunREG may represent a helpful tool to reconcile transcriptome and proteome data.

Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of protective antigen (PrAg), edema factor (EF), and lethal factor (LF). To intoxicate host mammalian cells, PrAg, the cell-binding moiety of the toxin, binds to cells and is then proteolytically activated by furin on the cell surface, resulting in the active heptameric form of PrAg. This heptamer serves as a protein-conducting channel that translocates EF and LF, the two enzymatic moieties of the toxin, into the cytosol of the cells where they exert cytotoxic effects. The anthrax toxin delivery system has been well characterized. The amino-terminal PrAg-binding domain of LF (residues 1-254, LFn) is sufficient to allow translocation of fused "passenger" polypeptides, such as the ADP-ribosylation domain of Pseudomonas exotoxin A, to the cytosol of the cells in a PrAg-dependent process. The protease specificity of the anthrax toxin delivery system can also be reengineered by replacing the furin cleavage target sequence of PrAg with other protease substrate sequences. PrAg-U2 is such a PrAg variant, one that is selectively activated by urokinase plasminogen activator (uPA). The uPA-dependent proteolytic activation of PrAg-U2 on the cell surface is readily detected by western blotting analysis of cell lysates in vitro, or cell or animal death in vivo. Here, we describe the use of PrAg-U2 as a molecular reporter tool to test the controversial question of what components are required for uPAR-mediated cell surface pro-uPA activation. The results demonstrate that both uPAR and plasminogen play critical roles in pro-uPA activation both in vitro and in vivo.

Histone chaperones are a group of histone-interacting proteins, involved in several important cellular functions. These chaperones are essential to facilitate ordered assembly of nucleosomes, both in replication dependent and independent manner. Replication independent function of histone chaperone is necessary for histone eviction during transcriptional initiation and elongation. In this chapter we have discussed a method to evaluate the role of histone chaperone NPM1 (the only known chaperone to get acetylated with functional consequence) in the transcriptional activation which is acetylation dependent.

The heat shock (HS) response is the major cellular defense mechanism against acute exposure to environmental stresses. The hallmark of the HS response, which is conserved in all eukaryotes, is the rapid and massive induction of expression of a set of cytoprotective genes. Most of the induction occurs at the level of transcription. The master regulator, heat shock transcription factor (HSF, or HSF1 in vertebrates), is responsible for the induction of HS gene transcription in response to elevated temperature. Under normal conditions HSF is present in the cell as an inactive monomer. During HS, HSF trimerizes and binds to a consensus sequence in the promoter of HS genes, stimulating their transcription by up to 200-fold. We have shown that a large, noncoding RNA, HSR1, and the translation elongation factor eEF1A form a complex with HSF during HS and are required for its activation.

Oligonucleotides with guanosine-rich (G-rich) sequences often have unusual physical and biological properties, including resistance to nucleases, enhanced cellular uptake, and high affinity for particular proteins. Furthermore, we have found that certain G-rich oligonucleotides (GROs) have antiproliferative activity against a range of cancer cells, while having minimal toxic effects on normal cells. We have investigated the mechanism of this activity and studied the relationship between oligonucleotide structural features and biological activity. Our results indicate that the antiproliferative effects of GROs depend on two properties: the ability to form quadruplex structures stabilized by G-quartets and binding affinity for nucleolin protein. Thus, it appears that the antiproliferative GROs are acting as nucleolin aptamers. Because nucleolin is expressed at high levels on the surface of cancer cells, where it mediates the endocytosis of various ligands, it seems likely that nucleolin-dependent uptake of GROs plays a role in their activity. One of the GROs that we have developed, a 26-nucleotide phosphodiester oligodeoxynucleotide now named AS1411 (formerly AGRO100 or GRO26B-OH), is currently being tested as an anticancer agent in Phase II clinical trials.

The telomeric poly(ADP-ribose) polymerase (PARP), tankyrase 1, modulates the impact of telomerase inhibition on human cancer cells. Thus, overexpression of tankyrase 1 in telomerase-positive cancer cells confers resistance to telomerase inhibitors, such as MST-312, whereas pharmacological inhibition of tankyrase 1 enhances telomere shortening by MST-312. These facts indicate that tankyrase 1 could be a target for telomere-directed molecular cancer therapy. Here, the authors describe a convenient method to monitor the telomeric function of tankyrase 1. This protocol takes much less time than the telomere Southern blot analysis and can be utilized as a rapid screening system for tankyrase 1 inhibitors that are effective in intact cells. For direct monitoring of tankyrase 1 PARP activity, a protocol for the in vitro enzyme assay is also described.

Ribonucleic acid (RNA) import into mitochondria occurs in a variety of organisms. In mammalian cells, several small RNAs are imported in a natural manner; transfer RNAs (tRNAs) can be imported in an artificial way, following expression of corresponding genes from another organism (yeast) in the nucleus. We describe how to establish and to analyze such import mechanisms in cultured human cells. In detail, we describe (1) the construction of plasmids expressing importable yeast tRNA derivatives in human cells, (2) the procedure of transfection of either immortalized cybrid cell lines or primary patient's fibroblasts and downregulation of tRNA expression directed by small interfering RNA (siRNA) as a way to demonstrate the effect of import in vivo, (3) the methods of mitochondrial RNA isolation from the transfectants, and (4) approaches for quantification of RNA mitochondrial import.

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.

The development of in vitro assays to analyze pre-mRNA splicing resulted in the discovery of many fundamental features characterizing splicing signals and the machinery that completes this process. Because in vitro assays can be manipulated by various biochemical approaches, the versatility of investigating alternative pre-mRNA splicing in the test tube appears endless. Importantly, modifications in reaction conditions can lead to the accumulation, isolation, and characterization of reaction intermediates, a prerequisite for gaining mechanistic insights into how the spliceosome carries out intron removal, and how regulatory elements assist the general splicing machinery in defining splice sites and alternative exons. These considerable experimental advantages have made the in vitro splicing system a standard assay, even though this approach is independent from RNA transcription and other RNA processing events, and in some respects deviates from the natural process of mRNA biogenesis. Here, we describe the tools and techniques necessary to carry out in vitro splicing assays. Analyses of various experimental designs are presented to highlight the approaches taken to gain insights into the mechanisms by which splice site recognition and activation are communicated with the general splicing machinery. Methods to measure the kinetics of splicing, to observe the formation of the pre-spliceosomal complexes, and to manipulate and modify the in vitro system to resolve the regulatory influences in alternative splicing are presented.

The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis in cells, and defects in this pathway are associated with cancer and neurodegenerative diseases. To assess 26S proteasome function in living animals, we developed a ubiquitin-luciferase reporter for bioluminescence imaging. The reporter was degraded rapidly under steady-state conditions and stabilized in a dose- and time-dependent manner in response to proteasome inhibitors. Using bioluminescence imaging after one dose of the chemo-therapeutic proteasome inhibitor bortezomib (PS-341), proteasome function in tumor xenografts was blocked within 30 min and returned to nearly baseline by 46 h. After a 2-week regimen of bortezomib, however, imaging of target tumors showed significantly enhanced proteasome inhibition that no longer returned to baseline. The ubiquitin-luciferase reporter enables repetitive tissue-specific analysis of 26S proteasome activity in vivo and should facilitate development and validation of proteasome inhibitors in mouse models, as well as investigations of the ubiquitin-proteasome pathway in disease pathogenesis.

Protein N-methylation has recently emerged as a major mechanism involved in regulating gene transcription and epigenetic inheritance. Transcriptional coactivators and corepressors have both been shown to associate with protein N-methyltransferases. These enzymes methylate arginine or lysine residues within histones as well as other proteins, such as transcription factors. Here, we discuss the recent advances in our understanding of protein methylation and its involvement in gene expression. Protocols are presented for studying transcriptionally relevant protein methylation.

In vitro analysis of transcription and the factors that play a role in transcription require preparation of an extract that faithfully reproduces in vivo transcription. This unit describes protocols for generating extracts of mammalian cells and Drosophila embryos that are competent for in vitro transcription reactions using naked DNA or chromatin templates and for primer extension analysis of transcripts.

A HeLa cell extract can be used as an alternative to rabbit reticulocyte lysate (RRL) for in vitro translation of mRNAs. It is particularly suited for synthesis of proteins that cannot be translated by RRL, is more cap dependent, has a higher capacity for faithful initiation at the correct start codon, and is more likely representative of translation in intact mammalian cells. This unit provides a detailed protocol for production and use of an mRNA-dependent cell-free translation system from HeLa extract. A support protocol describes production and purification of the mRNA.