Figure 2.  The Bright Particle and Its Localization Are ASH1 3'UTR-Dependent

(A) Particle formation and localization of the GFP-MS2 fusion protein (green) in cells (K699) that express the ASH1 reporter (lacZ-MS2-ASH1 3'UTR). Probes to lacZ used for FISH (red) colocalized (yellow) with the particle. 68% of the cells with GFP signal had a bright, single particle, and 78% of the cells with signal had localized particles. The DAPI signal is to the right of each panel. All samples were fixed.

(B) Diffuse signal from the GFP-MS2 fusion protein in cells (K699) that express the ADHII reporter (lacZ -MS2-ADHII 3'UTR). Probes to lacZ used for FISH (red) colocalized (yellow) with the diffuse GFP signal (green). 4% of the cells with GFP signal had a single, bright particle of equivalent intensity to those found in cells containing the ASH1 reporter and 2% of cells with signal had localized particles.

(C) The GFP-MS2 fusion expressed without the chimeric RNA reporter. 0% of the cells had a bright, single particle and 0% were localized.

(D) The GFP-MS2 expressed with a reporter that does not contain the MS2-binding sites (lacZ-ASH1 3'UTR). FISH shows the localization of the lacZ containing RNA. 8% of the cells with GFP signal had a single, bright particle equivalent in intensity to those containing the ASH1 reporter RNA and 0% of these were localized, even though the reporter without the MS2-binding sites still localized.

(E) Cell expressing the ASH1 reporter (left) simultaneously used for FISH for the endogenous ASH1 mRNA (red, right). Images were deconvolved to increase the sensitivity of detection and six adjacent planes of the restored image volume superimposed. GFP signal, not resolvable in unrestored images (e.g., A-D and other figures), was detectable using this approach.

(F) A cell prepared as in E, except the endogenous ASH1 mRNA signal (red) and ASH1 reporter signal (green) are colocalized (yellow). The transcription sites of the endogenous ASH1 RNA in the nucleus can be seen (yellow). Bar, 5 µm.