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RNA binding properties of the R3H domain of poly(A)-specific ribonuclease (PARN)
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 harbors, in addition to a well characterized RNA-recognition motif (RRM), another putative RNA-binding motif: the R3H-domain. R3H-domains have previously been found in a diverse range of proteins involved in single stranded nucleic acid binding suggesting that the domain is a nucleic acid interacting motif. However, its RNA binding properties has not been experimentally verified. Here, we show that the R3H domain of PARN binds poly(A) and poly(U). Therefore, our study verifies that the R3H domain binds RNA and suggests that the R3H-domain of PARN could play a role in recognizing poly(A).

Keyword [en]
mRNA degradation, deadenylation, Poly (A)-specific ribonuclease, poly(A)-specificity, R3H-domain
Identifiers
URN: urn:nbn:se:uu:diva-100499OAI: oai:DiVA.org:uu-100499DiVA: diva2:210396
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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