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

Che Chuan Yang, Yi Ting Wang, Yu-Yuan Hsiao, Lyudmila G. Doudeva, Pan Hsien Kuo, Sih Yao Chow, Hanna S. Yuan

Research output: Contribution to journalArticle

5 Scopus citations

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.

Original languageEnglish
Pages (from-to)1748-1759
Number of pages12
JournalRNA
Volume16
Issue number9
DOIs
StatePublished - 1 Jan 2010

Keywords

  • Apoptotic nuclease
  • Crystal structure
  • DNA degradation
  • DNase
  • RNA turnover
  • RNase
  • RNase PH

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