Fluorescence Correlation Spectroscopy (FCS) provides insights into mRNA binding proteins
Proteins are critical for countless roles in our bodies including structure, function, and regulation of our tissues and organs. To create proteins from the genes in our DNA, an intermediate molecule, mRNA, is needed. mRNA is made from DNA in the nucleus and then transported out of the nucleus where it is translated into protein.
Dr. Finn Cilius Nielsen and his team at the Center for Genomic Medicine, Denmark, work to understand the molecules which control mRNAs. Dr. Nielsen’s lab recently published an article using superresolution microscopy, fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS, respectively) to better understand these molecules.
We spoke with Àngels Mateu-Regué, a graduate student in Dr. Nielsen’s lab and lead author on the publication, about their findings and use of this technique.
Can you briefly describe your area of research and the findings in your article?
mRNAs are coated with a wide-range of proteins, forming complexes known as messenger ribonucleoprotein particles or “mRNPs”. These proteins regulate mRNA transport, translation, stability, etc. It had been hypothesized that mRNAs could be transported in groups, thus coordinating transport and translation of different mRNAs in space and time.
To answer these questions, we used a combination of superresolution microscopy and fluorescence correlation spectroscopy (FCS). Using these techniques, we could see that mRNAs exist as single molecules in cells and that the association between different RNA-binding proteins happen via mRNA-dependent interactions. Therefore, coordinate protein synthesis is unlikely to be a result of assembly of different mRNAs in the same mRNP granule.
How did fluorescence correlation spectroscopy (FCS) contribute to your findings?
We characterized the dynamic behavior of the mRNA-protein complexes using two GFP-tagged RNA binding proteins present in these particles: IMP1 and YBX1. Through the analysis of their autocorrelation curves, we learned that mRNP granule dynamics is not described by a simple diffusion model. We could conclude that mRNP granules diffuse in at least two different motions in live cells. Data visualization and quantification of diffusion was very easy to perform. GFP alone, in contrast, gave a very fast and simple 3D diffusion compared to the two other factors.
Additionally, we were also able to count the number of proteins bound to a complex by comparing the brightness of monomeric GFP with the brightness of the complex.
How did you use Fluorescence Cross-Correlation Spectroscopy (FCCS)?
We wanted to study the nature of the interaction between the above mentioned proteins: IMP1 and YBX1. Specifically, we wanted to see whether the interaction was direct or RNA-mediated (both proteins bound to the same mRNA). This time, we used a mCherry-tagged YBX1 and we co-transfected cells with GFP-IMP1 or a GFP-IMP1 mutant that is not able to bind to RNA. It was very clear to see that when we used wild type GFP-IMP1 and mCherry-YBX1, fluorescence signals coming from GFP or mCherry were synchronous, and that resulted in a positive cross-correlation curve. On the other hand, when we used the GFP-IMP1 mutant, we could clearly see that the cross-correlation curve was completely flat, indicating that the interaction between the two proteins is RNA-dependent.