Figure 1: Use of Spinach RNA aptamer to monitor localization of mRNAs in S. cerevisiae.

(a) Spinach tagging does not alter the function of tagged GAL1 transcript. Fivefold serial dilutions of strains grown on the indicated media. (b) Expression of GAL1 Spinach transcripts. Expression of tagged and untagged GAL1 transcripts were analysed by RT–qPCR in cells grown overnight in raffinose and shifted to galactose for 1 or 2 h. Expression analysis was performed in three replicates. (c–e) Localization of GAL1 Spinach transcripts. After overnight culture in raffinose at 30 °C, cells were grown for 2 h in galactose (c) or in glucose (d) and incubated with indicated concentration of DFHBI. (e) Cells grown in galactose for 2 h and incubated with 100 μΜ DFHBI were washed and observed just after DFHBI removal. Scale bar, 10 μm. (f) Localization of ASH1 mRNAs scored by Spinach tagging or FISH. Examples of ASH1 Spinach transcripts localization at different stages of the cell cycle. ASH1 transcripts are visible as dots (arrows) in the nucleus or the cytoplasm of the cells and Nup159–mCherry reveals the nuclear periphery (left panel). Histogram showing the distribution of ASH1 transcripts analysed using Spinach tagging or FISH in wt or nup60Δ dividing cells (right panel, Spinach tagging analysis was performed in three replicates with n=68 for wt and n=58 for nup60Δ cells; FISH analysis was performed in two replicates with n=67 for wt and n=92 for nup60Δ cells). Scale bar, 10 μm. (g) Deletion of NUP60 promotes nuclear retention of ASH1 transcripts in the mother cell. Nuclear accumulation was analysed by Spinach tagging (left panel), and quantified and compared with FISH analysis (right panel). Spinach tagging analysis was performed in three replicates with n=356 for wt and n=369 for nup60Δ cells; FISH analysis was performed in two replicates with n=222 for wt and n=400 for nup60Δ cells. Scale bar, 2 μm. Error bars show s.d. Results have been compared using a Student’s t-test, ***P<0.001,**0.001<P<0.01, *0.01<P<0.05. Image acquisition of Spinach-tagged transcripts was done with exposure set at 200 ms with 30% of a 50 mW laser. Saturation of the signal was observed for exposure set above 300 ms.