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. 2015 Oct 16;6:8670. doi: 10.1038/ncomms9670

Figure 4. Ubiquitination of CNOT7 by MEX-3C is required for its deadenylation activity.

Figure 4

CNOT7 is ubiquitinated in vivo in a MEX-3C-dependent manner. Ubiquitin blots following denaturing immunoprecipitation (IP) of endogenous (a) or overexpressed (b) CNOT7. IB, immunoblot; EV, empty vector (Supplementary Fig. 3a,b shows expression/loading controls). (c) Identification of the lysine residues in CNOT7 ubiquitinated by MEX-3C in vivo. 4K-mutant: K196R,K200R,K203R,K206R. (d) Ubiquitination of CNOT7 by MEX-3C is required for its deadenylation activity. Deadenylation assay was performed as described in Fig. 3. *P<0.05; unpaired Student's t-test. (e) CNOT7 4K-mutant rescues MEX-3C-mediated degradation of FF-Luc-HLA-A2 3′UTR reporter mRNA in-vivo. FF-Luc-HLA-A2-3′UTR mRNA levels were analysed as in Fig. 2. CNOT7 catalytic inactive mutant (Inact): D40A/E42A. *P<0.05 and **P<0.005, both versus wtCNOT7+ MEX-3C treatment; unpaired Student's t-test. Results are expressed as mean±s.d. of three independent experiments and relative to EV control (Supplementary Fig. 4c shows expression/loading controls for this figure).