FIGURE 4:

Overexpression of Y14 prevents the decay of a reporter mRNA. (A) HeLa tet-off cells were cotransfected with vectors expressing the TNFα-3′ UTR–containing βG reporter (diagram) and the reference GFP and the mock or FLAG-protein expression vector for 24 h. After doxycycline addition for 0, 1, 2, or 4 h, total RNA was prepared from each transfectant and then subjected to an RNase protection assay using ³²P-labeled probes (rightmost lane) specific to the βG or GFP mRNA. Top, a representative autoradiogram of the RNase protection assay. (B) The mRNA degradation assay was performed as in A with the effectors Magoh (lanes 4–6) and Y14 (∆C and full-length, lanes 7–12). Immunoblotting in both A and B was performed using anti-FLAG. M, DNA markers. (C) The semilogarithmic graph shows the rate of βG mRNA decay in each transfectant. Each percentage was calculated by dividing the βG level (normalized to the GFP level) of each time point to that of time zero. The t_1/2 was calculated using the function ln(0.5)/b, where b is the slope of the trend line obtained from the equation y = ne^−bx. The results were obtained from three independent experiments; however, SD was invisible in the logarithmic scale.