In Situ Study of Interactions between Endogenous c-myc mRNA with CRDBP in a Single Living Cell by Combining Fluorescence Cross-Correlation Spectroscopy with Molecular Beacons.

mRNA-protein interactions play key roles in facilitating various biological functions in gene expression regulations and even the progression of diseases. However, it is still a challenge to directly monitor mRNA-protein interactions in a single living cell at present. Herein, we propose a new strategy for real-time studying of mRNA-protein interactions in a single living cell using fluorescence cross-correlation spectroscopy (FCCS) and molecular beacon (MB) labeling techniques. The c-myc mRNA and coding region determinant binding protein (CRDBP) were used as models. We first evaluated the performances of unmodified (2'-deoxy) and modified (2'-O-methyl) MBs and found that the 2'-O-methyl loop MB (2'-O-methyl loop domain, 2'-deoxy stem region) has high affinity to target mRNA and good nuclease resistance. Then we constructed stable cell line expressing mCherry-CRDBP using lentivirus infection, and on the basis of FCCS, we established an efficient method for quantifying the interaction of c-myc mRNA with CRDBP in a single living cell. The RNA binding domains of CRDBP cover two RNA recognition motifs (RRM) and four K homologies (KH). Furthermore, we constructed the truncated variants and point mutants on RNA binding domains of CRDBP, systematically studied the effects of RNA binding domains of CRDBP on c-myc mRNA-CRDBP interaction in living cells, and found that KH3-4 is indispensable for c-myc mRNA binding, KH1-2 plays a supplementary role, and RRM1-2 shows no binding ability to c-myc mRNA. Our work reveals the mechanisms of c-myc mRNA-CRDBP interactions and provides a general strategy for quantifying the interactions of endogenous mRNA with protein in a single living cell.