Biochemistry

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

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

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.

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

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.

During development, cells must quickly switch from one cell state to the next to execute precise and timely differentiation. One method to ensure fast transitions in cell states is by controlling gene expression at the post-transcriptional level through action of RNA-binding proteins on mRNAs. The ability to accurately identify the RNA targets of RNA-binding proteins at specific stages is key to understanding the functional role of RNA-binding proteins during development. Here we describe an adapted formaldehyde RNA immunoprecipitation (fRIP) protocol to identify the in vivo RNA targets of a cytoplasmic RNA-binding protein, YTHDC2, from testis, during the first wave of spermatogenesis, at the stage when germ cells are shutting off the proliferative program and initiating terminal differentiation (Bailey et al., 2017). This protocol enables quick and efficient identification of endogenous RNAs bound to an RNA-binding protein, and facilitates the monitoring of stage-specific changes during development.

To determine the molecular and functional interactions between RNA-binding proteins (RBPs) and their targets RNAs, is of fundamental importance to understand the dynamic organization of the nervous system in health and disease. Nevertheless, this task has remained elusive due to the lack of specific protocols and experimental systems that would allow the combination of biochemical analysis with in vivo functional genetics. In this manuscript, we describe a trustworthy and detailed methodology to establish the molecular organization and intracellular function of RBPs/RNA multimeric complexes in a cell type-defined manner by using the powerful GAL4/UAS system for gene expression in Drosophila melanogaster.

Graphic abstract:

Immunoprecipitation for protein-RNA interaction in Drosophila.

RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein.

The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. Enriched RNA classes and the nucleotide frequency in these RNAs inform on the intrinsic specificity of the recombinant protein. The simple and versatile protocol can be adapted to other RNA binding proteins and total RNA libraries from any cell type or tissue.

Graphic abstract:


Figure 1. Schematic of the in vitro RNA immunoprecipitation (vitRIP) protocol

Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex to post-transcriptionally silence host target genes. We hereby describe the methodological details of how we recovered cross kingdom sRNA effectors of the oomycete pathogen Hyaloperonospora arabidopsidis during infection of its host plant Arabidopsis thaliana. This Bio-protocol contains two parts: first, a detailed description on the procedure of plant AGO/sRNA co-immunopurification and sRNA recovery for Illumina high throughput sequencing analysis. Second, we explain how to perform bioinformatics analysis of sRNA sequence reads using a Galaxy server. In principle, this protocol is suitable to investigate AGO-bound sRNAs from diverse host plants and plant-interacting (micro)organisms.