mRNA Interactome Capture

Talented scientists, sophisticated equipment, and advanced technologies make Creative Biolabs a leading service provider in RNA-protein interaction analysis by mRNA interactome capture.

What Is mRNA Interactome Capture?

RNA-binding proteins (RBPs) are a group of proteins exhibiting essential functions in gene expressions, such as RNA processing, mRNA localization, post-transcriptional modification, and translation. By binding to specific RNA targets, RBPs participate in regulating a variety of biological processes. Screening and identification of these RNA-protein interactions are necessary to reveal specific function mechanisms.

mRNA interactome capture (RIC) is a recently developed method for extensive and unbiased identification of active RBPs in living cells, which allows systematically capturing all RNA-protein interactions that occur in the transcriptome at a specific time. This method identifies RBPs on a global-wide level in a sample, and it can be utilized in combination with other specific assays (e.g., crosslinking and immunoprecipitation step (CLIP)) and analytic tools (e.g., RNA sequencing, mass spectrometry (MS)). So far, RIC has already been a powerful tool in RBPs screening and identification in several different types of samples (including mammalian, plants, yeast, and embryonic cell). It also can be used to proliferate cells, monitor the dynamic responses of RNA-protein interactions in different biological states.

Fig.1 Overview of RNA interactome capture. (Castello, 2013)

Advantages of mRNA Interactome Capture

RIA usually starts with irradiation of living cells with ultraviolet (UV) light at 254 nm to crosslink the photoreactive nucleotide bases with amino acids generating covalent bonds between RNA and proteins. Also, this cross-link can be strengthened by the photoactivatable-ribonucleoside-enhanced cross-linking (PAR-CL) protocol, which is achieved through the incorporation of the photoactivatable nucleotide 4-thiouridine (4SU) into nascent RNA and irradiation with UV light at 365 nm. Subsequently, cells are lysed and polyadenylated RNA is purified and captured with oligo(dT) magnetic beads. The RNA-protein complexes are washed stringently. The RBPs are released after RNase treatment and analyzed by MS.

RIC displays several favorable advantages over other mRNA-protein interaction analytic approaches:

• UV irradiation promotes RNA-protein cross-link efficiently rather than protein-protein interactions;
• Physiological protein-RNA interactions are "fixed" by direct UV irradiation;
• Oligo(dT) capture is highly stable in high salt and denaturing agents allowing stringent purification;
• Effective capture and analysis of all interactome.

Fig.2 Schematic representation of RNA interactome capture. (Castello, 2015)

Choose RIC Services, Choose Creative Biolabs

With an accumulation of experience and expertise in RNA analysis and services, Creative Biolabs is dedicated to offering a full range of RNA-protein interaction analysis services, especially by the method of RNA interactome capture. Additionally, other approaches for rapid RNA-protein interaction analysis are also within our abilities, such as chromatin isolation by RNA purification (ChIRP), RNA immunoprecipitation (RIP), and cross-linking and immunoprecipitation (CLIP).

As a business leader, we are glad and proud to provide flexible and reliable RNA-based services to accelerate your research. So, if you have any questions or special requirements, please feel free to contact us for more information.

Reference

1. Castello, A.; et al. System-wide identification of RNA-binding proteins by interactome capture. Nature protocols. 2013, 8(3): 491.
2. Castello, A. The emerging universe of RNA-binding proteins. The Biochemist. 2015, 37(2): 33-38.