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Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease
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.
University of Texas-Houston Medical School, Houston.
Institute of Molecular and Cell Biology, Proteos, Singapore.
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2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 1, 163-170 p.Article in journal (Refereed) Published
Abstract [en]

Poly(A)-specific ribonuclease (PARN) is a mammalian 3′-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), ∼10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.

Place, publisher, year, edition, pages
2010. Vol. 285, no 1, 163-170 p.
Keyword [en]
mRNA deadenylation, poly(A)-specific ribonuclease, active site, trinucleotides, poly(A)-specificity, poly(U)-specificity
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-100494DOI: 10.1074/jbc.M109.043893ISI: 000273070100017PubMedID: 19901024OAI: oai:DiVA.org:uu-100494DiVA: diva2:210379
Available from: 2009-04-01 Created: 2009-04-01 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Poly(A)-specific Ribonuclease (PARN): Structural and Functional Studies of Poly(A) Recognition and Degradation
Open this publication in new window or tab >>Poly(A)-specific Ribonuclease (PARN): Structural and Functional Studies of Poly(A) Recognition and Degradation
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Regulation of mRNA degradation is a powerful way for the cell to regulate gene expression. A critical step in eukaryotic mRNA degradation is the removal of the poly(A) tail at the 3'-end of the mRNA. Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. In addition to the exonuclease domain, PARN harbors two RNA-binding domains, a classical RNA recognition motif (RRM) and an R3H-domain. In this project we have studied mechanisms by which PARN specifically recognizes and degrades poly(A).

We investigated the RNA binding properties of PARN by using electrophoretic mobility shift assays and filter-binding analysis and we could show that PARN binds poly(A) with high affinity and specificity. Furthermore, we showed that the RRM and R3H domains of PARN separately could bind to poly(A).

To investigate specificity for and recognition of poly(A) in the active site of PARN, we performed a kinetic analysis on a repertoire of trinucleotide substrates in vitro. We showed that PARN harbors affinity for adenosines in the active site and that both the penultimate and the 3' end located nucleotide play an important role for providing adenosine-specificity in the active site of PARN.

Moreover, we solved a crystal structure of PARN in complex with m7GpppG cap analogue and showed that the cap binding and active sites overlap both structurally and functionally. By mutational analysis we identified residues in the active site that specifically recognize adenosines. Furthermore, biochemical data showed that the adenosine specificity in the active site is lost when Mn2+ is used instead of Mg2+ as divalent metal ion.

Taken together, these results demonstrate that both RNA-binding properties of the RRM and R3H-domains in addition to base recognition in the active site contributes to PARN poly(A)-specificity.

 

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 629
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-98710 (URN)978-91-554-7484-3 (ISBN)
Public defence
2009-05-08, BMC B41, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-04-17 Created: 2009-03-02 Last updated: 2010-12-16Bibliographically approved

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