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

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 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.

The exozyme model: A continuum of functionally distinct complexes

“…Third, new work has shown that Ce, Os, and Hs Rrp46 can form stable homodimers with species-specific RNase and DNase activities ( Fig. 3; Yang et al 2010). These findings expand upon previous observations showing a role for a subset of exosome subunits (Rrp6 and Rrp46) in the apoptotic degradosome of Ce Xue 2003, 2006).…”

“…Indirect exosome subunit-RNA interactions via additional proteins such as KSRP and TTP are also postulated by the exozyme model (Chen et al 2001;Gherzi et al 2004;Chou et al 2006). Finally, Rrp46 homodimers with DNA binding and DNase activity show that exozymes have RNA-independent functions (Yang et al 2010). As the exozyme model is both testable and consistent with all the previously published data, it should be examined and rigorously tested to determine if it is a viable explanation of exosome subunit function.…”

“…Finally, exosome subunits could form smaller exozymes that carry out the RNA processing and turnover functions of the complex. One such exozyme, already mentioned, is the Rrp46 homodimer (Yang et al 2010).…”

“…Human Dis3 has also been shown to have 28S rRNA processing functions that are distinct from three other exosome subunits (Slomovic et al 2010). Finally, Rrp46 homodimers have been shown to bind and degrade RNA in vitro (Yang et al 2010). In sum, certain RNA processing and surveillance functions attributed to the entire exosome complex could be formed independently by individual exosome subunits or subsets of exosome subunits.…”

“…An exosome-like complex is evolutionarily conserved in all domains of life, with a bacterial analog (PNPase), nine-subunit forms in some archaea, and nine-to 11-subunit complexes found in all eukaryotes studied to date (Table 1; Koonin et al 2001;Evguenieva-Hackenberg et al 2003;YehudaiResheff et al 2003;Schmid and Jensen 2008). Our understanding of exosome complexes is derived from a compilation of data from in vitro and in silico approaches coupled with genetic and biochemical evidence from several model organisms including Saccharomyces cerevisiae (Sc), Sulfolobus solfataricus (Ss), Archaeoglobus fulgidus (Afu), Schizosaccharomyces pombe (Sp), Trypanosoma brucei (Tb), Arabidopsis thaliana (At), Oryza sativa (Os), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), and Homo sapiens (Hs) tissue culture cells (Mitchell et al 1997;Briggs et al 1998;Allmang et al 1999aAllmang et al ,b, 2000Brouwer et al 2001;Chen et al 2001;Estevez et al 2001Estevez et al , 2003Andrulis et al 2002;Brouwer et al 2002;Butler 2002;Evguenieva-Hackenburg et al 2003;Forler et al 2003;Buttner et al 2005;Vanacova and Stefl 2007;Yang et al 2010).…”