The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).
Despite important methodological advances made in the past few years, a widely applicable, cost-effective and easily scalable procedure that can be routinely used to isolate ribonucleoprotein complexes (RNPs) remains elusive. We describe a versatile method that connects aspects of existing methods in a workflow optimized to reach the above goals and called it Silica-based Acidic Phase Separation (SAPS)-capture. To validate the method, the 18S rRNP of S. cerevisiae was captured. To illustrate its applicability, we isolated a repertoire of RNPs in A. thaliana. This procedure can provide the community with a powerful tool to advance the study of ribonomes and RNPs in any organism or tissue type.
Although methodological advances have been made over the past years, a widely applicable, easily scalable and cost-effective procedure that can be routinely used to isolate specific ribonucleoprotein complexes (RNPs) remains elusive. We describe the “Silica-based Acidic Phase Separation (SAPS)-capture” workflow. This versatile method combines previously described techniques in a cost-effective, optimal and widely applicable protocol. The specific RNP isolation procedure is performed on a pre-purified RNP sample instead of cell lysate. This combination of protocols results in an increased RNP/bead ratio and by consequence a reduced experimental cost. To validate the method, the 18S rRNP of S. cerevisiae was captured and to illustrate its applicability we isolated the complete repertoire of RNPs in A. thaliana. The procedure we describe can provide the community with a powerful tool to advance the study of the ribonome of a specific RNA molecule in any organism or tissue type.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.