Identification of the Active Site of Poly(A)-specific Ribonuclease by Site-directed Mutagenesis and Fe2+-mediated Cleavage

Abstract

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. 2؉ binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3-exonuclease domain of Escherichia coli DNA polymerase I. Poly(A)-specific ribonuclease (PARN)1 is the only mammalian poly(A)-specific 3Ј-exoribonuclease identified and characterized thus far (1-5). It has been shown that PARN is an oligomeric, highly processive, and mRNA cap-interacting exonuclease (3, 6 -8). A reaction pathway for PARN degradation has been proposed, and key characteristics of this pathway are the release of 5Ј-AMP as the mononucleotide product and the requirement for a 3Ј located adenosine residue with a free 3Ј-hydroxyl group (5). PARN has been cloned from human and Xenopus laevis, and the PARN polypeptide is M r 74,000 in both cases (2, 9). However, the active mammalian enzyme is significantly larger due to its oligomeric structure and most likely consists of three identical subunits (3).The function of PARN in vivo is still unknown. However, compelling evidence suggests that PARN is a key nuclease involved in both mRNA decay (7) and mRNA poly(A) tail length control during X. laevis oocyte development (2, 9). The cap interacting and poly(A) degrading properties of PARN also provide evidence that PARN may play a role in controlling initiation of protein synthesis (3, 6 -8). Besides a possible role in controlling translation, the mRNA cap binding property of PARN has a direct role in stimulating PARN degradation efficiency (3, 6, 7) and in amplifying the processive mode of degradation (8). We have recently proposed a simple model for how PARN operates (8). In this model, oligomeric PARN interacts simultaneously with the mRNA 3Ј-end-located poly(A) tail and the 5Ј-end-located cap structure. It has been suggested, based on kinetic evidence, that the cap-binding site is separate from the active site of PARN (8).The amino acid sequence of PARN implies that PARN belongs to the RNase D family of nucleases (2,11,12), of which the 3Ј-exonuclease (or proofreading) domain of Escherichia coli DNA polymerase

Coordination of Divalent Metal Ions in the Active Site of Poly(A)-specific Ribonuclease

“…Some activity could be recovered from the PARN(D382C) mutant after several hours of incubation, although we never managed to detect any hydrolytic activity when using the other cysteine-substituted PARN mutants (data not shown). This suggests that all these conserved acidic amino acid residues are essential for PARN activity, in keeping with our previous results (13).…”

“…However, it remains to be shown that the glutamic acid residue Glu-30 coordinates a divalent metal ion, although available data show that Glu-30 is essential for PARN activity (Fig. 3) and important for the interaction of divalent metal ions (13). Most likely the introduction of a cysteine residue at this position severely affects both the divalent metal ion coordination properties, caused by the replacement of an oxygen atom by sulfur, and the configuration of the PARN active site, because cysteine lacks one methylene group in its side chain compared with glutamate, making it impossible to rescue this particular mutant by replacing Mg 2ϩ with a soft divalent metal ion.…”

“…Furthermore, Fe 2ϩ -mediated hydroxyl radical cleavage provided evidence that Asp-28, Glu-30, and Asp-382, were required for coordination of catalytically important metal ions (13). Fig.…”

“…shown that four conserved acidic amino acid residues Asp-28, Glu-30, Asp-292, and Asp-382 of PARN are essential for the catalytic activity of PARN (13). Furthermore, Fe 2ϩ -mediated hydroxyl radical cleavage provided evidence that Asp-28, Glu-30, and Asp-382, were required for coordination of catalytically important metal ions (13).…”

“…Site-directed mutagenesis has shown that these conserved residues are essential for catalytic activity and that they are required for the binding of divalent metal ions to PARN (13). Based on these observations it was proposed (13) that PARN utilizes the twometal ion mechanism for its catalysis, as suggested for the Escherichia coli 3Ј-exonuclease domain of DNA polymerase I (pol I) (14,15), although the stoichiometry of divalent metal ion binding in the active site of PARN is not yet known. To fully understand the catalytic mechanism of PARN it is of fundamental importance to determine metal ion binding coordination and stoichiometry in its active site.…”