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The mRNA Cap Structure Stimulates Rate of Poly(A) Removal and Amplifies Processivity of Degradation
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2001 In: J. Biol. Chem, Vol. 276, 27923-27929 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2001. Vol. 276, 27923-27929 p.
Identifiers
URN: urn:nbn:se:uu:diva-97046OAI: oai:DiVA.org:uu-97046DiVA: diva2:171826
Available from: 2008-04-18 Created: 2008-04-18Bibliographically approved
In thesis
1. Allosteric Regulation of mRNA Metabolism: -Mechanisms of Cap-Dependent Regulation of Poly(A)-specific Ribonuclease (PARN)
Open this publication in new window or tab >>Allosteric Regulation of mRNA Metabolism: -Mechanisms of Cap-Dependent Regulation of Poly(A)-specific Ribonuclease (PARN)
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3’ end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5’ end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using site-directed mutagenesis and fluorescence spectroscopy. We identified tryptophan 475, located within the RNA recognition motif (RRM) of PARN, as crucial for cap binding. A crystal structure of PARN bound to cap revealed that cap binding is mediated by the nuclease domain and the RRM of PARN. Tryptophan 475 binds the inverted 7-Me-guanosine residue through a stacking interaction. Involvement of the nuclease domain in cap binding suggests that the cap site and the active site overlap. Mutational analysis showed that indeed amino acids involved in cap binding are crucial for hydrolytic activity of PARN. Taken together, we show that PARN is an allosteric enzyme that is activated by the cap structure and that the allosteric cap binding site in one PARN subunit corresponds to the active site in the other PARN subunit.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 423
Keyword
Cell and molecular biology, mRNA degradation, deadenylation, cap, poly(A), PARN, allosteric regulation, Cell- och molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-8647 (URN)978-91-554-7163-7 (ISBN)
Public defence
2008-05-09, B42, Biomedicinskt centrum, Husargatan 3, Uppsala, 10:00
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Available from: 2008-04-18 Created: 2008-04-18Bibliographically approved

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