Figure 5.

mRNAs translation in CoFe granules is required for localization

(A) Schematic of TRICK reporter system. Ribosomes on translated RNAs “knock off” the PP7-CP-GFP fusion, whereas on untranslated RNAs the coat protein remains bound.

(B) z-stacked images of TRICK-tagged mRNAs coexpressing the MS2-CP-mCh fusion and the PP7-CP-GFP fusion, in + and – glucose. Scale bars: 3 μm.

(C) Quantification of MS2-CP-mCh-only granules as a percentage of total granules observed in TRICK-tagged mRNAs in + and – glucose conditions. Error bars are ±SD.

(D) Schematic of PDC1 premature stop codon (sc) and stem loop (sl) insertion.

(E) z-stacked images of cells expressing Dcp2p-CFP- and PDC1-MS2-tagged mRNA. PDC1-MS2 (sc) has a premature stop codon in the ORF.

(F) z-stacked images of strains expressing Dcp2p-CFP with pPDC1-MS2 or pPDC1-MS2 (sl). pPDC1-MS2 (sl) has a stem loop upstream of the ORF.

(G) Scatterplot of mRNA granules per cell in PDC1-MS2-tagged mRNA with or without a premature stop codon and in strains bearing pPDC1-MS2 with or without the stem loop. Error bars are ±SD. Scale bars: 2 μm.

(H) Relative fold change of (1) PDC1 MS2-tagged mRNA relative to untagged PDC1 mRNA, (2) PDC1-MS2 mRNA in strains harboring a premature stop codon (sc) relative to a strain without, (3) PDC1-MS2 mRNA on a plasmid (pPDC1-MS2 mRNA) relative to genomic PDC1-MS2 mRNA, and finally (4) pPDC1-MS2 mRNA in strain with a stem loop upstream of the ORF relative to a pPDC1-MS2 mRNA without a stem loop. Error bars are ±SD.