Fluorescence Correlation and Cross-Correlation Spectroscopy Unveil Cytoplasmic mRNP Composition and Dynamics

Abstract

Understanding the mRNA life cycle requires analysis of the dynamic macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study mRNP complexes because they readily provide information about the molecular composition, stoichiometry, heterogeneity and dynamics of the particles. We developed FCS protocols for analysis of live cells and cellular lysates, and demonstrate the feasibility of analysing common cytoplasmic mRNPs composed of core factor YBX1, IMPs (or IGF2BPs) and their interactions with other RNA binding proteins such as PABPC1, ELAVL2 (HuB), STAU1 and FMRP. FCCS corroborated previously reported RNA dependent interactions between the factors and provided an estimate of the relative overlap between the factors in the mRNPs. In this way FCS and FCCS provide a new and useful approach for the quantitative and dynamic analysis of mRNP macromolecular complexes that may complement current biochemical approaches.