Figure 5.

Presence of a boxC motif enhances Tob1 APRO ability to stimulate deadenylation in cellulo

A) Reverse transcription polymerase chain reaction (RT-PCR) amplification of the poly(A) tails of the β-globin reporter transcript. HEK293 Tet-Off cells were co-transfected with plasmids expressing the β-globin reporter and the different HA- and GFP-tagged BTG/Tob fusion proteins as indicated (or empty GFP expression vector). Fragments comprising ∼200 bp of the 3′ UTR of the β-globin mRNA in addition to poly(A) tails were amplified using a RACE-PAT assay. Experiments were repeated twice with, in each case, two biological duplicates. B) Profiles of the poly(A) tail lengths of the β-globin reporter. The gel presented in A was quantified using ImageJ and relative average profiles were plotted after adjusting the maximal signal intensity to 100% for each lane. C) Poly(A) tail lengths were analysed and presented as box plots indicating weighted mean, weighted upper and lower quartile, and weighted 5–95% distribution. Significant p values (*: p < 0.05; ** p < 0.01) from ANOVA tests between corresponding wild-type and mutant proteins are indicated. D) A western blot analysis of the cell lysates corresponding to the transfections performed in A is shown.