Structural and biochemical characterization of CRN-5 and Rrp46: an exosome component participating in apoptotic DNA degradation.
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 A and 2.0 A, 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.
Yang
Che-Chuan
CC
Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, 10617 Taiwan, Republic of China.
Wang
Yi-Ting
YT
Hsiao
Yu-Yuan
YY
Doudeva
Lyudmila G
LG
Kuo
Pan-Hsien
PH
Chow
Sih Yao
SY
Yuan
Hanna S
HS
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D000954:Antigens, Surface / Q000737:chemistry* / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D018360:Crystallography, X-Ray; D053938:DNA Fragmentation; D005095:Exoribonucleases / Q000737:chemistry* / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D016601:RNA-Binding Proteins; D016415:Sequence Alignment
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; C552049:CRN-5 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; C467564:EXOSC5 protein, human; D016601:RNA-Binding Proteins; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex