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- Henriksson, Niklas, 1976-
(author)
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Poly(A)-specific Ribonuclease (PARN) : Structural and Functional Studies of Poly(A) Recognition and Degradation
- 2009
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Doctoral thesis (other academic/artistic)abstract
- 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.
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- Nilsson, Per, et al.
(author)
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A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties
- 2007
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:45, s. 32902-32911
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Journal article (peer-reviewed)abstract
- 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.
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- Souter, Petra, et al.
(author)
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Patterns of genetic structuring in the coral Pocillopora damicornis on reefs in East Africa.
- 2009
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In: BMC Ecology. - : Springer Science and Business Media LLC. - 1472-6785. ; 9, s. 19-
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Journal article (peer-reviewed)abstract
- This study showed that population differentiation in P. damicornis varied over spatial scales and that this variability occurred at both evolutionary and ecological time scales. This paradox is discussed in light of stochastic recruitment and small scale population structures found in other species of coral. The study also identifies potential source reefs, such as those within Mnemba Conservation area near Zanzibar and genetically isolated reefs such as those within Malindi Marine National Park and Reserve in northern Kenya.
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- Wu, Mousheng, et al.
(author)
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Structural basis of m(7)GpppG binding to poly(A)-specific ribonuclease
- 2009
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In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 17:2, s. 276-286
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Journal article (peer-reviewed)abstract
- Poly(A)-specific ribonuclease (PARN) is a homodimeric, processive, and cap-interacting 3' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. The crystal structure of a C-terminally truncated PARN in complex with m(7)GpppG reveals that, in one subunit, m(7)GpppG binds to a cavity formed by the RRM domain and the nuclease domain, whereas in the other subunit, it binds almost exclusively to the RRM domain. Importantly, our structural and competition data show that the cap-binding site overlaps with the active site in the nuclease domain. Mutational analysis demonstrates that residues involved in m(7)G recognition are crucial for cap-stimulated deadenylation activity, and those involved in both cap and poly(A) binding are important for catalysis. A modeled PARN, which shows that the RRM domain from one subunit and the R3H domain from the other subunit enclose the active site, provides a structural foundation for further studies to elucidate the mechanism of PARN-mediated deadenylation.
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