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A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties
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.
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 45, 32902-32911 p.Article in journal (Refereed) Published
Abstract [en]

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.

Place, publisher, year, edition, pages
2007. Vol. 282, no 45, 32902-32911 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97049DOI: 10.1074/jbc.M702375200ISI: 000250625400039PubMedID: 17785461OAI: oai:DiVA.org:uu-97049DiVA: diva2:171829
Available from: 2008-04-18 Created: 2008-04-18 Last updated: 2017-12-14Bibliographically 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
Opponent
Supervisors
Available from: 2008-04-18 Created: 2008-04-18Bibliographically approved
2. 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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