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A 54-kDa fragment of the Poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting Poly(A)-specific 3' exonuclease.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
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2000 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 31, 24222-24230 p.Article in journal (Refereed) Published
Abstract [en]

We have previously identified a HeLa cell 3' exonuclease specific for degrading poly(A) tails ofmRNAs, Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated witha similar 3' exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specificribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180-220 kDa, Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits.The purified nuclease activity released 5'-AMP as the reaction product and degraded poly(A) in a highlyprocessive fashion. The activity required monovalent cations and was dependent on divalent metal ions. TheRNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3' end-located poly(A) was degraded by the activity. RNA substrates capped with m(7)G(5')ppp(5')G were more efficiently degraded than noncapped RNA substrates. Addition of free m7G(5')ppp(5')G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5' end cap structure andpoly(A) degradation at the 3' end of the RNA.

Place, publisher, year, edition, pages
2000. Vol. 275, no 31, 24222-24230 p.
Keyword [en]
Adenosine Monophosphate/metabolism, Animals, Cattle, Chromatography; Affinity/methods, Exoribonucleases/isolation & purification/*metabolism, Movement, Peptide Fragments/metabolism, Protein Binding, Protein Structure; Quaternary, RNA Caps/*metabolism, RNA Processing; Post-Transcriptional, Substrate Specificity, Thymus Gland/*enzymology
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-16139DOI: 10.1074/jbc.M001705200ISI: 000088564200109PubMedID: 10801819OAI: oai:DiVA.org:uu-16139DiVA: diva2:43910

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2008-05-17 Created: 2008-05-17 Last updated: 2013-09-17Bibliographically approved
In thesis
1. Poly(A)-Specific Ribonuclease (PARN)
Open this publication in new window or tab >>Poly(A)-Specific Ribonuclease (PARN)
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Degradation of the mRNA 3'-end located poly(A) tail is an important step for mRNA decay in mammalian cells. Thus, to understand mRNA decay in detail, it is important to identify the catalytic activities involved in degrading poly(A). We identified and purified a 54-kDa polypeptide responsible for poly(A)-specific 3' exonuclease activity in calf thymus extracts. The 54-kDa polypeptide is a proteolytic fragment of the poly(A)-specific ribonuclease (PARN) 74-kDa polypeptide. PARN is a divalent metal ion dependent, poly(A)-specific, oligomeric, processive and cap interacting 3' exonuclease. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. The interaction with the 5'-end cap structure stimulates PARN activity and also amplifies the processivity of the deadenylation reaction. Furthermore, the cap binding site and the active site of PARN are separate from each other. To characterise the active site of PARN, we per-formed side-directed mutagenesis, Fe2+-mediated hydroxyl radical cleavage and metal ion switch experiments. We have demonstrated that the conserved acidic amino acid residues D28, E30, D292 and D382 of human PARN are essential for PARN activity and that these amino acid residues are directly involved in the co-ordination of at least two metal ions in the active site of PARN. Phosphorothioate modification on RNA substrates revealed that the pro-R oxygen atom of the scissile phosphate group interacts directly with the metal ion(s). Based on our studies, we propose a model for the action of PARN. Similarly to what has been observed for ribozymes, aminoglycoside antibiotics inhibit PARN activity, most likely by the displacement of catalytically important divalent metal ions. Among the aminoglycoside antibiotics tested, neomycin B is the most potent inhibitor. We speculate that inhibition of enzymes using similar catalytic mechanisms as PARN could be a reason for the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2001. 29 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 677
Cell and molecular biology, Poly(A)-specific ribonuclease, PARN, deadenylation, processive, oligomeric, cap interacting, aminoglycoside, active site, FE2+-mediated cleavage, metal ion switch, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
urn:nbn:se:uu:diva-1590 (URN)91-554-5194-2 (ISBN)
Public defence
2002-01-18, lecture hall C10: 305 at the Biomedical Center (BMC), Uppsala, 09:15
Available from: 2001-12-11 Created: 2001-12-11 Last updated: 2013-09-17Bibliographically approved

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