Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

Figure 3

Proteolytic cleavage of AUF1-. (a) Time-course cleavage pattern of AUF1- during 48 h at room temperature. The protein was cleaved into two small fragments (~10 kDa) after 18 h. Each of the time points is indicated above the gel. “M” and “0” indicate the low-range size marker (Bio-Rad) and the protein not incubated at room temperature (control), respectively. (b) Time-course cleavage pattern of AUF1- in the presence of 0.2 mM PMSF during 5 days at room temperature. Each of the time points by hours is indicated above the gel. “M” and “0” indicate the low-range size marker (Bio-Rad) and the protein not incubated at room temperature (control), respectively. (c) Limited proteolysis of AUF1- by trypsin in a concentration-dependent manner at 10°C for 90 min. “M” and “C” indicate the marker as shown in (a) and the nontrypsin-treated control, respectively. Labels 1 to 5 represent the ratios of trypsin to AUF1- protein: label 1, 1/2000; label 2, 1/1500; label 3, 1/1000; label 4, 1/500; and label 5, 1/300. The possible RRM1 fragment is indicated by a white arrow head. (d) Limited proteolysis of AUF1- by subtilisin in a concentration-dependent manner at 4°C for 60 min. “M” and “C” indicate the marker as shown in (a) and the nonsubtilisin-treated control, respectively. Labels 1 to 4 represent the ratios of subtilisin to AUF1- protein: label 1, 1/2000; label 2, 1/1500; label 3, 1/1000; and label 4, 1/500. The possible RRM1 fragment is indicated by a white arrow head.

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