Abstract
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).
Highlights
Ribonucleoprotein complexes (RNPs), composed of both RNA molecule(s) and one or more RNA-binding proteins (RBPs), are known to play key roles in cell homeostasis and cell fate by controlling post-transcriptional gene regulation and RNA processing
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
Summary
Ribonucleoprotein complexes (RNPs), composed of both RNA molecule(s) and one or more RNA-binding proteins (RBPs), are known to play key roles in cell homeostasis and cell fate by controlling post-transcriptional gene regulation and RNA processing. Does each method have its own strengths and weaknesses, but there are some challenges, involving yield, cost and specificity, common to all Overcoming these could result in major advances in the field and a better characterization of multiple RNPs, even the ones difficult to study, such as those associated with low-copy-number RNAs. With the recent publication of organic phase separation protocols such as Cross-Linked RNA eXtraction (XRNAX) [21], orthogonal organic phase separation (OOPS) [22] and Phenol-Toluol extraction (PTex) [23], we address these challenges and propose future experimental strategies that build on these methods. Together with the fact that a lot is still unknown about the role and importance of numerous transient RNA–protein interactions, it is advisable to experimentally validate identified targets, no matter how stringent the purification procedure [23,24]
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