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

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

*Corresponding author for this work

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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.

Original languageEnglish
Article numbere119
JournalNucleic Acids Research
Volume49
Issue number20
Number of pages15
ISSN0305-1048
DOIs
Publication statusPublished - 2021

Bibliographical note

Publisher Copyright:
© 2021 The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

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