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


ABSTRACT: Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.

SUBMITTER: Mateu-Regue A 

PROVIDER: S-EPMC8599746 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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

Mateu-Regué Àngels À   Christiansen Jan J   Bagger Frederik Otzen FO   Hellriegel Christian C   Nielsen Finn Cilius FC  

Nucleic acids research 20211101 20


Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and re  ...[more]

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