Fig 2: The TTP CIM promotes mRNA decay cooperatively with conserved TTP tryptophans. (A) Representative Northern blots monitoring mRNA decay of ß-globin mRNA tethered to TTP-CTD wild-type (wt) or mutant proteins with the CIM deleted (?CIM), conserved tryptophans mutated to alanines (WA), or both (WA,?CIM). (B) Graph quantifying four repeats of mRNA decay assays in panel A. Error bars represent standard deviation. (C) Western blots monitoring expression levels of TTP-CTD fusion proteins in panel A. (D) Same as panel A, but with wild-type or mutant MS2-TTP full-length fusion proteins. (E) Graph quantifying four repeats of mRNA decay assays in panel D. (F) Western blots monitoring expression levels of TTP fusion proteins in panel D. (G) Western blots showing proteins co-immunoprecipitating (IP, right panels) with the indicated FLAG-tagged MS2-TTP-CTD fusion proteins from HEK293T cells after treatment with RNase A, as compared with input samples (left). IP samples correspond to 2.5% of the input. (H) Same as panel G, but monitoring proteins associated with FLAG-tagged MS2-TTP fusion proteins. *, p < 0.05; Student's two-tailed t-test.

Fig 3: TTP tetraproline motifs do not enhance TTP mRNA decay activity. (A) Representative Northern blots monitoring mRNA decay of ß-globin mRNA tethered to indicated MS2-TTP wild-type (wt) or mutant proteins with the tetraproline motifs mutated to serines (PS), the CIM deleted (?CIM), or both (PS,?CIM). (B) Graph quantifying three repeats of mRNA decay assays in panel A. Error bars represent standard deviation. (C) Western blots monitoring expression levels of TTP fusion proteins in panel A. (D) Same as panel A, with indicated mutant MS2-TTP fusion proteins with tetraproline motifs mutated to serines and conserved tryptophans to alanines (PS,WA), or additional deletion of the CIM (PS,WA,?CIM). (E) Graph quantifying four repeats of mRNA decay assays in panel D. (F) Western blots monitoring expression levels of TTP fusion proteins in panel D. (G) Bar-graph comparing half-lives of ß-globin mRNA tethered to the indicated MS2-TTP fusion proteins with or without mutation in tetraproline motifs (PS). Error bars represent standard deviation from 3 or 4 experiments. (H) Western blots showing proteins co-immunoprecipitating (IP, right panels) with indicated FLAG-tagged MS2-TTP fusion proteins from HEK293T cells after treatment with RNase A, as compared with input samples (left). IP samples correspond to 2.5% of the input. *, p < 0.05; n.s., p > 0.05; Student's two-tailed t-test.

Fig 4: TTP-mediated ARE-mRNA decay is highly dependent of the CIM in the presence of active MK2. (A) Representative Northern blots monitoring mRNA decay of β-globin mRNA containing an ARE from GM-CSF mRNA, in the presence of TTP wild-type (wt) or ΔCIM proteins, co-expressed with constitutive active (MK2A) or catalytic dead (MK2D) MK2 kinase. (B) Western blots monitoring expression levels of TTP and MK2 proteins in panel A. Note, FLAG-MS2-GFP migrates similarly to FLAG-MK2. (C) Graph quantifying three repeats of mRNA decay assays in panel A. Error bars represent standard deviation. *, p < 0.05; Student's two-tailed t-test.

Fig 5: TTP activity is highly dependent on an intact CIM in the presence of MK2. (A, C) Representative Northern blots (n = 3) monitoring degradation of ß-globin mRNA tethered to indicated MS2-TTP fusion proteins in the presence of constitutive active (MK2A) or catalytically dead (MK2D) MK2 kinase in HeLa tet-off cells. (B, D) Western blots monitoring expression levels of TTP fusion proteins in the experiments in panels A and C, respectively. *, p < 0.05; Student's two-tailed t-test.