Effects of Pan3 isoforms and their domain truncations on Pan2 deadenylase activity in vivo. NIH3T3 cells were transfected with a plasmid coding for HA-Pan2 alone or together with a plasmid coding for one of the HA-tagged WT or truncated Pan3 isoforms. β-Globin mRNA constitutively expressed (A) or transiently induced (B–D) in the same cells served as a reporter transcript. Total cytoplasmic RNA was extracted for Northern blot analysis to check poly(A) size distribution of the β-globin reporter mRNA (upper panels of A,B; upper left panels of C,D). Poly(A)– RNA samples were prepared with oligo(dT) and RNase H treatment. Levels of the indicated HA-tagged proteins were visualized by Western blot analysis (lower panels of A,B; lower left panels of C,D). The poly(A) size distribution profiles (middle panels of A,B; right panels of C,D) were obtained by scanning each lane of the Northern blots using ImageJ software. Red arrows (A,B) highlight changes of the poly(A) size distribution profile. Red and blue dashed lines (C,D) serve as a reference to compare the effects caused by different Pan3 domain truncations on the poly(A) size distribution.
