Protocols in Current Issue
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0 Q&A 252 Views Jul 5, 2023

Toxin–antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I–VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods.

Key features

• Detailed protocol for the large-scale purification of ToxIN type III toxin–antitoxin complexes from E. coli.

• The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA.

• Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones.

• The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.

Graphical overview

0 Q&A 418 Views Jul 5, 2023

In vitro translation systems are a useful biochemical tool to research translational regulation. Although the preparation of translation-competent cell extracts from mammals has often been a challenge, the commercially available rabbit reticulocyte lysate (RRL) is an exception. However, its valid use, investigating the mechanism of translation machinery such as ribosomes in RRL, presents an analytic hurdle. To overcome this issue, the hybrid translation system, which is based on the supplementation of purified human ribosomes into ribosome-depleted RRL, has been developed. Here, we describe the step-by-step protocol of this system to study translation driven by ribosomes lacking post-translational modifications of the ribosomal protein. Moreover, we combined this approach with a previously developed reporter mRNA to assess the processivity of translation elongation. This protocol could be used to study the potency of heterologous ribosomes.

0 Q&A 1541 Views Jun 5, 2023

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins’ (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins.

Key features

• Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution.

• iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells.

• iCLIP facilitates the identification of sequence motifs recognized by RBPs.

• Allows quantitative analysis of genome-wide changes in protein-RNA interactions.

• Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples.

Graphical overview

0 Q&A 433 Views Dec 20, 2022

Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2’-O-methyl (2’OMe), 2’-fluoro (2’F), pseudouridine (ᴪ), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases.

0 Q&A 1284 Views Dec 20, 2022

The importance of studying the mechanistic aspects of long non-coding RNAs is being increasingly emphasized as more and more regulatory RNAs are being discovered. Non-coding RNA sequences directly associate with generic RNA-binding proteins as well as specific proteins, which cooperate in the downstream functions of the RNA and can also be dysregulated in various physiologic states and/or diseases. While current methods exist for identifying RNA–protein interactions, these methods require high quantities of input cells or use pooled capture reagents that may increase non-specific binding. We have developed a method to efficiently capture specific RNAs using less than one million input cells. One single oligonucleotide is used to pull down the target RNA of choice and oligonucleotide selection is driven by sequence accessibility. We perform thermal elution to specifically elute the target RNA and its associated proteins, which are identified by mass spectrometry. Ultimately, two target and control oligonucleotides are used to create an enrichment map of interacting proteins of interest.

Graphical abstract

Schematic representation of the SOCRATES workflow. SOCRATES utilizes a single 20-mer oligonucleotide for RNA pull down followed by a temperature elution series and liquid chromatography–mass spectrometry (LC-MS)/MS to identify specific RNA–protein interactions.

0 Q&A 1860 Views Dec 5, 2022

RNA is a vital component of the cell and is involved in a diverse range of cellular processes through a variety of functions. However, many of these functions cannot be performed without interactions with proteins. There are currently several techniques used to study protein–RNA interactions, such as electrophoretic mobility shift assay, fluorescence anisotropy, and filter binding. RNA-pulldown is a technique that uses biotinylated RNA probes to capture protein–RNA complexes of interest. First, the RNA probe and a recombinant protein are incubated to allow the in vitro interaction to occur. The fraction of bound protein is then captured by a biotin pull-down using streptavidin-agarose beads, followed by elution and immunoblotting for the recombinant protein with a His-tag–reactive probe. Overall, this method does not require specialized equipment outside what is typically found in a modern molecular laboratory and easily facilitates the maintenance of an RNase-free environment.

Graphical abstract

0 Q&A 1286 Views Aug 5, 2022

In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle.

Graphical abstract:

Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.

0 Q&A 2736 Views May 5, 2022

Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as well as between cell populations, contributing to the diversity of mammalian tissues. Earlier methods that analyzed gene expression at the level of translation, such as polysomal- or ribosomal-profiling, required large amounts of starting material to isolate enough RNA for analysis by microarray or RNA-sequencing. Thus, rare or less abundant cell types within tissues were not able to be properly studied with these methods. Translating ribosome affinity purification (TRAP) utilizes the incorporation of an eGFP-affinity tag on the large ribosome subunit, driven by expression of cell-type specific Cre-lox promoters, to allow for identification and capture of transcripts from actively translating ribosomes in a cell-specific manner. As a result, TRAP offers a unique opportunity to evaluate the entire mRNA translation profile within a specific cell type, and increase our understanding regarding the cellular complexity of mammalian tissues.

Graphical abstract:

Schematic demonstrating TRAP protocol for identifying ribosome-bound transcripts specifically within cerebellar Purkinje cells.

0 Q&A 1476 Views Apr 5, 2022

In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring the lengths of single-stranded RNA separated through denaturing urea polyacrylamide gel electrophoresis (urea PAGE), which are visualized by a label-free fluorescence SYBR Gold, and quantified in a multi-function imager. This label-free method is easy to conduct, has low cost, and lacks the hazard of the traditional radio-labeled method. This method can also be adapted to the other Dicers and small RNAs.

0 Q&A 2248 Views Feb 20, 2022

Transfer RNAs (tRNAs) are highly abundant species and, along their biosynthetic and functional path, they establish interactions with a plethora of proteins. The high number of nucleobase modifications in tRNAs renders conventional RNA quantification approaches unsuitable to study protein-tRNA interactions and their associated functional roles in the cell. We present an immunoprecipitation-based approach to quantify tRNA bound to its interacting protein partner(s). The tRNA-protein complexes are immunoprecipitated from cells or tissues and tRNAs are identified by northern blot and quantified by tRNA-specific fluorescent labeling. The tRNA interacting protein is quantified by an automated western blot and the tRNA amount is presented per unit of the interacting protein. We tested the approach to quantify tRNAGly associated with mutant glycyl-tRNA-synthetase implicated in Charcot-Marie-Tooth disease. This simple and versatile protocol can be easily adapted to any other tRNA binding proteins.

Graphic abstract:

Figure 1. Schematic of the tRNA-Immunoprecipitation approach.

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