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PMID:20660080

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Citation

Yang, CC, Wang, YT, Hsiao, YY, Doudeva, LG, Kuo, PH, Chow, SY and Yuan, HS (2010) Structural and biochemical characterization of CRN-5 and Rrp46: an exosome component participating in apoptotic DNA degradation. RNA 16:1748-59

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 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.

Links

PubMed PMC2924534 Online version:10.1261/rna.2180810

Keywords

Amino Acid Sequence; Animals; Antigens, Neoplasm/chemistry; Antigens, Neoplasm/metabolism; Antigens, Surface/chemistry; Antigens, Surface/metabolism; Caenorhabditis elegans/enzymology; Caenorhabditis elegans Proteins/chemistry; Caenorhabditis elegans Proteins/metabolism; Carrier Proteins/chemistry; Carrier Proteins/metabolism; Cell Line; Crystallography, X-Ray; DNA Fragmentation; Exoribonucleases/chemistry; Exoribonucleases/metabolism; Humans; Models, Molecular; Molecular Sequence Data; Oryza sativa/enzymology; Sequence Alignment

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CAEEL:G5EG59

enables

GO:0003690: double-stranded DNA binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CAEEL:G5EG59

involved_in

GO:0032077: positive regulation of deoxyribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

CAEEL:G5EG59

enables

GO:0019899: enzyme binding

ECO:0000353: physical interaction evidence used in manual assertion

WB:WBGene00000797

F

Seeded From UniProt

complete

CAEEL:CRN4

enables

GO:0004536: deoxyribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ORYSJ:EXOS5

enables

GO:0003690: double-stranded DNA binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ORYSJ:EXOS5

enables

GO:0003727: single-stranded RNA binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ORYSJ:EXOS5

enables

GO:0004536: deoxyribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ORYSJ:EXOS5

enables

GO:0004540: ribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ORYSJ:EXOS5

enables

GO:0042802: identical protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q84T68

F

Seeded From UniProt

complete


See also

References

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