Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates
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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.
I: Nucleic Acids Research, Bind 49, Nr. 20, e119, 2021.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates
AU - Mateu-Regué, Àngels
AU - Christiansen, Jan
AU - Bagger, Frederik Otzen
AU - Hellriegel, Christian
AU - Nielsen, Finn Cilius
N1 - Publisher Copyright: © 2021 The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
U2 - 10.1093/nar/gkab751
DO - 10.1093/nar/gkab751
M3 - Journal article
C2 - 34478550
AN - SCOPUS:85121184725
VL - 49
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 20
M1 - e119
ER -
ID: 288267963