Figure 6.
Most PDC1 and ENO2 mRNAs Are Associated with Polysomes and Localized to mRNA Granules
(A and B) Polysome fractionation and qRT-PCR analysis on RNA prepared from individual fractions across polysome gradients. Polysomes were analyzed as described in Experimental Procedures. Traces depicting the changes in A254 across the gradient from the yMK1577 (ENO2-MS2L) and yMK1586 (PDC1-MS2L) strains grown in YPD are shown. The 40S (small ribosomal subunit), 60S (large ribosomal subunit), 80S (monosome), and polysome peaks are labeled. Below the percentage of each mRNA present in the fractions collected from the polysome gradient is plotted. Blue represents RNA in polysomal regions, whereas red is from the subpolysomal regions of the gradient. The total percentage in polysomal and subpolysomal regions across three repeat experiments is also depicted.
(C and D) (C) Representative images depicting the strategy for quantitating the percentage of PDC1 and ENO2 mRNAs in granules. Fluorescence was measured for the whole cell and granules only (as defined within the yellow lines), and the percentage of each mRNA present in the granules was calculated and plotted in (D).
Scale bar, 1 μm. Errors bars are ±SE. See also Figure S4.