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.