FIGURE 6:

Phosphomimetic Y14 inhibits mRNA decay and binds to the mRNA degradation complex and spliced mRNA with high affinity. (A) The in vivo mRNA degradation assay and following RNase protection assay were performed as in Figure 4. HeLa tet-off cells were mock transfected (lanes 1–3) or transfected with the wild-type Y14 (lanes 4–6) or Y14SE (lanes 7–9) expression vector. Immunoblotting was performed using anti-FLAG. (B) HEK293 cells were transiently transfected with the mock vector or the expression vector encoding FLAG-Y14 or FLAG-Y14SE. Cell lysates were subjected to immunoprecipitation using anti-FLAG, followed by immunoblotting using antibodies against each indicated protein. (C) In vitro splicing of the PIP85a pre-mRNA was performed in nuclear extracts prepared from mock, FLAG-Y14-expressing, or FLAG-Y14SE-expressing HEK293 cells (lanes 1–3). The reactions were subjected to immunoprecipitation using anti-FLAG; the resins were then washed with buffer containing 150 mM NaCl (lanes 4–6) or 300 mM NaCl (lanes 7–9). RNA was then extracted from the resins and fractioned on a denaturing gel, followed by autoradiography. Immunoblotting was performed using anti-FLAG. M, DNA markers. From top to bottom the bands represent the following RNA species: unspliced pre-mRNA, lariat intron-exon 2, lariat intron, and ligated exons.