Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates

Mateu-Regué, Àngels; Christiansen, Jan; Bagger, Frederik Otzen; Hellriegel, Christian; Nielsen, Finn Cilius

doi:10.1093/nar/gkab751

Cited by 7 publications

(10 citation statements)

References 27 publications

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“…Despite these technical limitations, FCS serves as a valuable tool for analyzing molecular diffusion through the measurement of fluorescence intensity fluctuations and has found widespread use in the investigation of various biological systems, including MLOs and phase-separated bodies. 264 It has been used thus far, among other applications, to determine the composition of RNPs, 265 to analyze the diffusion and hybridization rates of different oligonucleotide populations, 266 and to measure mRNA dynamics in living neurons. 267…”

Section: Methods For Determining Rna Structures Involved In Molecular...mentioning

confidence: 99%

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

Zacco,

Broglia,

Kurihara

et al. 2024

Chem. Rev.

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This comprehensive Review delves into the chemical principles governing RNA-mediated crowding events, commonly referred to as granules or biological condensates. We explore the pivotal role played by RNA sequence, structure, and chemical modifications in these processes, uncovering their correlation with crowding phenomena under physiological conditions. Additionally, we investigate instances where crowding deviates from its intended function, leading to pathological consequences. By deepening our understanding of the delicate balance that governs molecular crowding driven by RNA and its implications for cellular homeostasis, we aim to shed light on this intriguing area of research. Our exploration extends to the methodologies employed to decipher the composition and structural intricacies of RNA granules, offering a comprehensive overview of the techniques used to characterize them, including relevant computational approaches. Through two detailed examples highlighting the significance of noncoding RNAs, NEAT1 and XIST, in the formation of phase-separated assemblies and their influence on the cellular landscape, we emphasize their crucial role in cellular organization and function. By elucidating the chemical underpinnings of RNAmediated molecular crowding, investigating the role of modifications, structures, and composition of RNA granules, and exploring both physiological and aberrant phase separation phenomena, this Review provides a multifaceted understanding of the intriguing world of RNA-mediated biological condensates.

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Section: Methods For Determining Rna Structures Involved In Molecular...mentioning

confidence: 99%

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

Zacco,

Broglia,

Kurihara

et al. 2024

Chem. Rev.

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show abstract

“…We recently developed a FCS and FCCS protocol for analysis of ribonucleoprotein (RNP) assemblies employing cell lysates that complements live cell readings (20). Obviously, lysates do not reconcile the spatial resolution of live cells, but they have less spatial constraints and provide optimal readings because they can be diluted, which allows you to discern even small changes in binding.…”

Section: Fccs Analyses Of Isolated Brca1 Ring and Brct Domain Variant...mentioning

confidence: 99%

“…The experimental autocorrelation curves were obtained and analysed in ZEN 2011 software (Zeiss). The fits of the different models to the experimental data were also done in ZEN 2011, using their in-built models as described (20).…”

Section: Fluorescence Correlation Spectroscopy (Fcs)mentioning

confidence: 99%

Functional Assessment of Protein Variants in Structured Domains by Fluorescence Cross-Correlation Spectroscopy

Mateu-Regué,

Mariani,

Bagger

et al. 2024

Preprint

Self Cite

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With the expanding catalogue of novel disease-genes, there is an increasing need to establish the clinical significance of potential disease-causing variants. Based on the idea that pathogenic variants in structured protein domains disturb folding and association with macromolecular assemblies, we employed Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS) to assess in vivo protein complex formation. Since the molecular underpinning of BRCA-associated breast and ovarian cancers is well defined and data from a recent genome editing screening allowed us to compare the binding data with a reliable functional HRD test, we examined the binding of BRCA1 to BARD1 and RBBP8, respectively. The results demonstrate that FCCS, whether applied to full-length BRCA1 in live cells or to isolated domains in cellular lysates, reliably identified pathogenic BRCA1 RING or BRCT variants. We moreover demonstrate the feasibility of employing FCCS for analysis of HNPCC-related factor MSH2 and MEN1 factor Menin variants in combination with DNA mismatch repair factor MSH6 and transcription factor JUND, respectively. Because the procedure can be completed within a clinically relevant time frame, FCCS is an appealing complement to current clinical procedures for classifying variants. Given its generic nature and design, the approach can be applied to a variety of monogenic diseases.

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“…More specifically, here we focus on techniques with surface‐immobilized biomolecules as these have been well established for use in the characterization of RNP complex assemblies within cellular extracts. Confocal techniques for solution measurements are, thus far, underutilized for experiments in cellular extracts (Currie et al, 2015; Mateu‐Regué et al, 2021).…”

Section: Combining Single Molecule Fluorescence Microscopy and Partia...mentioning

confidence: 99%

Utilizing functional cell‐free extracts to dissect ribonucleoprotein complex biology at single‐molecule resolution

et al. 2023

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Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts. This strategy enables elucidation of the interaction and kinetic behavior of specific fluorescently labeled biomolecules within RNPs under conditions that approximate native cellular environments. In this review, we describe single molecule fluorescence microcopy approaches that dissect RNP‐driven processes within cellular extracts, highlighting general strategies used in these methods. We further survey biological advances in the areas of pre‐mRNA splicing and transcription regulation that have been facilitated through this approach. Finally, we conclude with a summary of practical considerations for the implementation of the featured approaches to facilitate their broader future implementation in dissecting the mechanisms of RNP‐driven cellular processes.This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

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Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates

Cited by 7 publications

References 27 publications

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

RNA: The Unsuspected Conductor in the Orchestra of Macromolecular Crowding

Functional Assessment of Protein Variants in Structured Domains by Fluorescence Cross-Correlation Spectroscopy

Utilizing functional cell‐free extracts to dissect ribonucleoprotein complex biology at single‐molecule resolution

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