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. 2017 Feb;23(2):134–141. doi: 10.1261/rna.057786.116

FIGURE 3.

FIGURE 3.

(A) Tagging of FBA1 mRNA at 3′ end with 24xPP7SL alters its cellular localization, with 3′-end fragments of PP7SL enriching in PBs. smFISH, imaging, and image processing is performed as in Figure 1B using strains expressing DCP2-GFP and either FBA1 or FBA1-24xPP7SL. Fluorescent DNA probes target either the FBA1 (i) or PP7SL (ii) moieties (Supplemental Table S3). White arrows show example cells with large mRNA foci colocalizing with the nucleus. Scale bar, 5 µm. (B) Tagging of GFA1 mRNA at its 3′ end with 24xPP7SL alters its cellular localization, with 3′-end fragments of PP7SL enriching in PBs. smFISH, imaging, and image processing is performed as in Figure 1B using strains expressing DCP2-GFP and either GFA1 or GFA1-24xPP7SL. Fluorescent DNA probes target either the GFA1 (i) or PP7SL (ii) moieties (Supplemental Table S3). White arrows show example cells with large mRNA foci colocalizing with the nucleus. Scale bar, 5 µm. (C,D) Quantification of colocalization of PBs with mRNA in glucose starvation conditions shown in Figure 3A,B, respectively. Percentage depicts number of PBs colocalizing with mRNAs compared to total number of PBs. n = 3 biological replicates with each >800 counted PBs; bars depict SD. Quantification of colocalization is performed on all planes of a 3D stack image using the Colocalization Threshold tool in Fiji. (E,F) Quantification of colocalization of nuclear DNA with mRNA foci in glucose-rich and glucose starvation conditions shown in Figure 3A,B, respectively. Percentage depicts nuclear DNA (judged by DAPI signal) colocalizing with mRNAs compared to total number of nuclear DNA. n = 3 biological replicates with each >300 counted nuclei; bars depict SD. Quantification of colocalization is performed on all planes of a 3D stack image using the Colocalization Threshold tool in Fiji. Note that since GFA1 is a low abundant mRNA, we hardly observe any nuclear signal that was brighter than the signal of an individual transcript. We therefore adjusted our quantification to a lower threshold compared to our PGK1 or FBA1 nuclear foci analysis to account for all mRNA/DAPI colocalization events.