Using Molecular Beacons to Study Dispersal of mRNPs from the Gene Locus
Before leaving the site of transcription, newborn messenger RNAs (mRNAs) become associated with a number of different proteins. How these large messenger ribonucleoprotein (mRNP) complexes then move through the dense nucleoplasm to reach the nuclear periphery has been a fascinating question for the last few years. We have studied the mechanism of this process by tracking individual mRNPs in real time. We were able to track mRNPs at single-molecule resolution because we utilized mRNAs that were engineered to have a sequence motif repeated 96 times in their untranslated region. These mRNAs were visualized with the help of molecular beacons that were specific for the repeated sequence; the binding of 96 molecular beacons to each mRNA molecule rendered them so intensely fluorescent that they were visible as fine fluorescent spots that could be tracked by high-speed video microscopy. In this chapter, we describe the details of the construction of genes containing the tandem repeats, the integration of such genes into the genome of a cell line, the design and testing of molecular beacons, time-lapse imaging of mRNPs, and computer-aided generation and analysis of the tracks of the individual mRNPs. These methods will be useful for studies of other dynamic processes such as mRNA export, splicing, and decay.
KeywordsMolecular beacons Single particle tracking mRNA transport Nuclear viscosity Live-cell imaging
We thank Diana Y. Vargas and Arjun Raj for their contributions. This work was supported by the National Institutes of Health Grant GM-070357.
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