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Poly(A)-specific ribonuclease (PARN) loses specificity for recognition of adenosine residues in the presence of Mn2+
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.
(English)Manuscript (Other academic)
Abstract [en]

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. PARN is a member of the DEDD superfamily of exonucleases and is dependent on divalent metal ions for its activity. Here, we have investigated how PARN poly(A) specificity can be modulated by using Mn2+ instead of Mg2+ as the divalent metal ion. In the presence of Mn2+ PARN lost specificity for poly(A) degradation. By using homotrinucleotides as substrates we could demonstrate that the loss in specificity was an intrinsic property of the active site in the presence of Mn2+ and not caused by changes in the RNA binding properties of PARN. We conclude that Mn2+ can be used as a probe to understand mechanisms behind poly(A) specificity in the active site of PARN.

Keyword [en]
mRNA degradation, deadenylation, Poly (A)-specific ribonuclease, active site, poly(A)-specificity, Mn2+
Identifiers
URN: urn:nbn:se:uu:diva-100497OAI: oai:DiVA.org:uu-100497DiVA: diva2:210384
Available from: 2009-04-01 Created: 2009-04-01 Last updated: 2010-01-14
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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