FIGURE 6.

The nab2 F73D mutant displays genetic interactions with mRNA export mutants mex67-5 and yra1–8 and mex67-5 nab2 F73D cells show nuclear poly(A) RNA accumulation. A, mex67-5 nab2 F73D cells or yra1–8 nab2 F73D cells have a slow growth phenotype. NAB2Δ mex67-5 or NAB2Δ yra1–8 cells maintained by a NAB2 URA3 plasmid and containing NAB2, nab2 ΔN, nab2 F72D, or nab2 F73D LEU2 test plasmids were grown to saturation, serially diluted in 10-fold dilutions and spotted on control and 5-FOA plates. Cells were grown at 30 °C. B, growth curve analysis of mex67-5 nab2 F73D cells confirms that they grow more slowly than mex67-5 NAB2 cells. mex67-5 NAB2Δ cells carrying only NAB2, nab2 F72D, or nab2 F73D plasmids were grown to saturation, diluted, and their optical density was measured at A₆₀₀ for 40 h as described under “Experimental Procedures.” C, mex67-5 nab2 F73D cells show nuclear accumulation of poly(A) RNA. NAB2Δ mex67-5 cells carrying only NAB2, nab2 F72D, or nab2 F73D plasmids were grown in liquid culture at 30 °C and poly(A) RNA (Poly(A)) was visualized by FISH using an oligo(dT) probe as described under “Experimental Procedures.” Cells were stained with DAPI to visualize the position of the nucleus. Corresponding differential interference contrast (DIC) images are shown. FISH results were quantitated as described under “Experimental Procedures” to assess the mean percentage of cells with nuclear accumulation of poly(A) RNA within 10 fields of at least 43 cells each (indicated to the right of the DIC images). Variation in the percentage of cells with nuclear poly(A) RNA in each field of cells from the mean was assessed by calculating the standard deviation: mex67-5 NAB2, 18.6 ± 4.8%; mex67-5 nab2 F72D, 20.3 ± 6.0%; and mex67-5 nab2 F73D, 30.8 ± 6.9. Thus, mex67-5 nab2 F73D cells exhibit a 1.7-fold increase in nuclear poly(A) RNA accumulation relative to mex67-5 NAB2 cells, whereas mex67-5 nab2 F72D cells show only a slight 1.1-fold increase. The percentages of nuclear poly(A) RNA accumulation observed in mex67-5 nab2 F73D cells were typical of multiple independent experiments.