EXPERIMENTAL FIGURE 10-37 Transport of mRNP particles from a yeast mother cell into the bud. (a) Yeast cells were engineered to express an ASH1 mRNA with binding sites for the bacteriophage λ N protein in its 5′ UTR, and an IST2 mRNA with binding sites for bacteriophage MS2 coat protein in its 3′ UTR. A fusion of green fluorescent protein to λ N protein (GFP-λN) and a fusion of red fluorescent protein to MS2 coat protein (RFP-MS2) were also expressed in the same cells. In other experiments, these fluorescently tagged sequence-specific RNA-binding proteins were shown to bind to their own specific binding sites engineered into the ASH1 and IST2 mRNAs, and not to each other’s binding sites. Both fluorescently tagged bacteriophage proteins also contained a nuclear localization signal so that those proteins that were not bound to their high-affinity binding sites in these mRNAs were transported into nuclei through nuclear pore complexes (see Chapter 13). This step was necessary to prevent high fluorescence from excess GFP-λN and RFP-MS2 in the cytoplasm. (b) Frames from a video of fluorescing cells. GFP-λN and RFP-MS2 were independently visualized by using millisecond alternating laser excitation of GFP and RFP. The nucleus next to the large vacuole in the mother cell near the center of each micrograph, as well as nuclei in neighboring cells, was observed by green and red fluorescence, as shown in the top and middle rows. A merge of the two images is shown in the bottom row, which also indicates the time elapsed between images. An RNP particle containing both the ASH1 mRNA with λN-binding sites and the IST2 mRNA with MS2-binding sites was observed in the mother-cell cytoplasm in the left column of images (arrow). The particle increased in intensity between 0.00 and 46.80 seconds, indicating that more of these mRNAs joined the RNP particle. The RNP particle was transported into the bud between 46.80 and 85.17 seconds and then became localized to the bud tip.

[Republished with permission of John Wiley & Sons, Inc., from Lange, S. et al., “Simultaneous transport of different localized mRNA species revealed by live-cell imaging,” 2008, Traffic, 9:(8)1256–67; permission conveyed through Copyright Clearance Center, Inc. See this paper to view the video.]