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

Abstract

Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the pres Poly(A)-specific ribonuclease (PARN)1 is a highly poly(A)-specific 3Ј-exonuclease that efficiently degrades mRNA poly(A) tails (1-6). PARN is oligomeric, most likely consisting of three subunits (i.e. homotrimer) (6), and interacts with both the 3Ј-end-located poly(A) tail and the 5Ј-end cap structure during degradation (6 -9). The interaction with the 5Ј-end cap structure enhances the rate of degradation (6, 7, 9) and amplifies the processivity of PARN activity (9). The functional significance of PARN is still unknown, although its high specificity for poly(A) and interaction with the cap structure provide strong arguments for PARN being involved in mammalian mRNA poly(A) tail degradation and therefore could play important roles in mRNA decay and initiation of protein synthesis (6 -9).PARN belongs to the DEDD family of nucleases, and four conserved acidic amino acids characteristic of this family are present in PARN (5, 10 -12). These amino acids are Asp-28, Glu-30, Asp-292, and Asp-382 in human PARN. 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.Structural analyses by x-ray or NMR are powerful strategies to reveal the presence of divalent metal ions in the active sites of enzymes. However, despite the high resolution provided by these techniques, these methods frequently fail to unambiguously locate catalytically important divalent metal ions. Furthermore, even if the divalent metal ions are correctly located in the active site by structural analysis, it is still necessary to functionally confirm the interaction between the divalent metal ions and their ligands (16). Thus, other techniques are required to locate functionally important metal ions. This is of particular importance when structural data, as in the case of PARN, is not available. Sulfur substitutions of oxygen believed to be in direct contact with Mg 2ϩ ions have successfully been used to locate ligands in direct con...

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…Fig. 7B also shows that the final product obtained in the presence of Mn 2ϩ ions was slightly smaller than that seen in the presence of Mg 2ϩ or Zn 2ϩ , suggesting that binding to different metal ions might affect the activity of the SARS-CoV ExoN by inducing subtle structural differences in the active site, as reported for several other nucleases (21,23). The data presented in Figs.…”

Global architecture of human poly(A)-specific ribonuclease by atomic force microscopy in liquid and dynamic light scattering

“…The interaction with the cap structure enhances efficiency of deadenylation [22][23][24] and amplifies the processivity of the enzymatic action [25]. PARN belongs to the RNase D family of nucleases [26] which is a part of the superfamily of DEDD nucleases [27] that are characterized by the presence of four conserved acidic amino acids (Asp, Glu, Asp, Asp) in the catalytic centre and require divalent metal ions for efficient catalysis [28,29]. Our recent work revealed that the active site of PARN per se harbors specificity for recognition of adenosine residues and that the nucleotide context around the scissile bond determines the efficiency of the hydrolysis [30].…”

Inhibition of human poly(A)-specific ribonuclease (PARN) by purine nucleotides: kinetic analysis

“…PARN also contains a RNA recognition motif (RRM), missing from the crystal structure, which harbours both poly(A) and cap binding properties [18] and stabilizes the overall structure of the enzyme [19]. The catalytic active site contains four invariant acidic amino acid residues, one glutamic acid and three aspartic acids (Asp28, Glu30, Asp292, and Asp382 in PARN) that coordinate catalytically essential divalent metal ions, such as Mg 2 þ [20,21], while His377 has been proposed to be essential for catalytic activity [17]. However, the crystal structure of the truncated PARN showed that no Mg(II) ions are harbored in the active site of the enzyme [17].…”