ABSTRACT
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.
Subject(s)
Ribonucleoproteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cost-Benefit Analysis , Ribonucleoproteins/metabolism , RNA/metabolismABSTRACT
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).
Subject(s)
Proteomics/methods , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , RNA/isolation & purification , RNA/metabolism , Ribonucleoproteins/isolation & purification , Ribonucleoproteins/metabolism , Animals , Humans , Protein Binding , Proteome/metabolism , RNA/genetics , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , TranscriptomeABSTRACT
BACKGROUND: Autotransporters represent a widespread family of secreted proteins in Gram-negative bacteria. Their seemingly easy secretion mechanism and modular structure make them interesting candidates for cell surface display of heterologous proteins. The most widely applied host organism for this purpose is Escherichia coli. Pseudomonas stutzeri A15 is an interesting candidate host for environmentally relevant biotechnological applications. With the recently characterized P. stutzeri A15 EstA autotransporter at hand, all tools for developing a surface display system for environmental use are available. More general, this system could serve as a case-study to test the broad applicability of autotransporter based surface display. RESULTS: Based on the P. stutzeri A15 EstA autotransporter ß-domain, a surface display expression module was constructed for use in P. stutzeri A15. Proof of concept of this module was presented by successful surface display of the original EstA passenger domain, which retained its full esterase activity. Almost all of the tested heterologous passenger domains however were not exposed at the cell surface of P. stutzeri A15, as assessed by whole cell proteinase K treatment. Only for a beta-lactamase protein, cell surface display in P. stutzeri A15 was comparable to presentation of the original EstA passenger domain. Development of expression modules based on the full-length EstA autotransporter did not resolve these problems. CONCLUSIONS: Since only one of the tested heterologous passenger proteins could be displayed at the cell surface of P. stutzeri A15 to a notable extent, our results indicate that the EstA autotransporter cannot be regarded as a broad spectrum cell surface display system in P. stutzeri A15.
