Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation |
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Authors: | Che-Chuan Yang Yi-Ting Wang Yu-Yuan Hsiao Lyudmila G. Doudeva Pan-Hsien Kuo Sih Yao Chow Hanna S. Yuan |
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Affiliation: | 1.Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, 10617 Taiwan, Republic of China;2.Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China;3.Taiwan International Graduate Program, Institute of Chemical Biology and Molecular Biophysics, Academia Sinica, Taipei 11529, Taiwan, Republic of China;4.Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China |
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Abstract: | Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 3′ processing of RNA during RNA turnover and surveillance. The Rrp46 homolog, CRN-5, was subsequently characterized as a cell death-related nuclease, participating in DNA fragmentation during apoptosis in Caenorhabditis elegans. Here we report the crystal structures of CRN-5 and rice Rrp46 (oRrp46) at a resolution of 3.9 Å and 2.0 Å, respectively. We found that recombinant human Rrp46 (hRrp46), oRrp46, and CRN-5 are homodimers, and that endogenous hRrp46 and oRrp46 also form homodimers in a cellular environment, in addition to their association with a protein complex. Dimeric oRrp46 had both phosphorolytic RNase and hydrolytic DNase activities, whereas hRrp46 and CRN-5 bound to DNA without detectable nuclease activity. Site-directed mutagenesis in oRrp46 abolished either its DNase (E160Q) or RNase (K75E/Q76E) activities, confirming the critical importance of these residues in catalysis or substrate binding. Moreover, CRN-5 directly interacted with the apoptotic nuclease CRN-4 and enhanced the DNase activity of CRN-4, suggesting that CRN-5 cooperates with CRN-4 in apoptotic DNA degradation. Taken together all these results strongly suggest that Rrp46 forms a homodimer separately from exosome complexes and, depending on species, is either a structural or catalytic component of the machinery that cleaves DNA during apoptosis. |
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Keywords: | RNA turnover DNA degradation crystal structure RNase PH RNase DNase apoptotic nuclease |
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