FIGURE 2.

Tethered TNRC6A stimulates decapping and the degradation of mRNA from the 5′ end, independent of translation and a poly(A) tail. A, schematic drawing of reporter genes used in Fig. 2. The line represents non-translated regions. All reporter genes contain the 3′-UTR region of PGK1, in which two tandem MS2 binding sites were inserted. B, tethering of TNRC6A stimulates 5′-to-3′ mRNA decay independent of a poly(A) tail. Wild type, ski2Δ, and xrn1Δ cells containing the indicated reporter gene and plasmids were grown, RNA samples were purified at each time point, and the reporter mRNA was analyzed by Northern blotting using a DIG-labeled PGK1 3′-UTR probe. C, TNRC6A reduces mRNA levels independently of translation and a poly(A) tail. Wild type cells harboring the indicated No-AUG reporter genes were grown, and RNA samples were analyzed by Northern blotting using a DIG-labeled MPT4 probe. The data represent the means of three independent experiments with S.D. values. D and E, tethering of the Mid domain of TNRC6A promotes 5′-to-3′ mRNA decay independent of translation and a poly(A) tail in a cap-dependent manner. Wild type, ski2Δ, and xrn1Δ cells harboring No-AUG-MS2-Rz (D) and Rz-No-AUG-MS2-Rz (E) reporter gene and plasmids were grown, and RNA samples were analyzed by Northern blotting using a DIG-labeled PGK1 3′-UTR probe. The half-lives of mRNA in the indicated cells are shown as the mean values of three independent experiments with S.D. values.