2016 Volume 31 Issue 2 Pages 110-118
Imaging of mRNAs in living cells is an effective approach to elucidate the molecular mechanism of biomolecules and cellular functions. Here we introduce methods to fluorescently label a specific mRNA using antisense oligonucleotide probes or RNA binding proteins. Next, we introduce examples of imaging dynamics of mRNAs in living cells. The first example is the analysis of dynamics of mRNAs in stress granules(SGs). mRNAs in COS7 cells were labeled with a poly(U)22 2'-O-methyl RNA and SGs were formed by arsenite. The analysis of mRNA dynamics using FRAP showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)+ mRNAs. The second example is single-molecule imaging of β-actin mRNAs in a chicken fibroblast. β-Actin mRNAs were labeled with MS2-GFP and their movement was analyzed. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA(DMACRO) at the leading edge was 0.3μm2/s. On the other hand, DMACRO in the perinuclear region was 0.02μm2/s. The destruction of microfilaments with cytochalasin D led to an increase in DMACRO to 0.2μm2/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of β-actin mRNA.
