Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation

CHE-CHUAN YANG,1,2 YI-TING WANG,2,3,4 YU-YUAN HSIAO,2,4 LYUDMILA G. DOUDEVA,2
PAN-HSIEN KUO,2,4 SIH YAO CHOW,2 and HANNA S. YUAN1,2,3
1Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, 10617 Taiwan, Republic of China
2Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
3Taiwan International Graduate Program, Institute of Chemical Biology and Molecular Biophysics, Academia Sinica, Taipei 11529, Taiwan,
Republic of China
4Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, Republic of China

ABSTRACT
Rrp46 was first identified as a protein component of the eukaryotic exosome, a protein complex involved in 39 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.
Keywords: RNA turnover; DNA degradation; crystal structure; RNase PH; RNase; DNase; apoptotic nuclease