In vitro deadenylation of mammalian mRNA by a HeLa cell 3′ exonuclease.

126

We have previously identified a HeLa cell 3 exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3 exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180 -220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3 end-located poly(A) was degraded by the activity. RNA substrates capped with m 7 G(5)ppp(5)G were more efficiently degraded than noncapped RNA substrates. Addition of free m 7 G(5)ppp(5)G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5 end cap structure and poly(A) degradation at the 3 end of the RNA.

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confidence: 76%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

A 54-kDa Fragment of the Poly(A)-specific Ribonuclease Is an Oligomeric, Processive, and Cap-interacting Poly(A)-specific 3′ Exonuclease

Martı́nez¹,

Ren²,

Thuresson³

et al. 2000

126

“…1 is the only mammalian poly(A)-specific 3Ј-exoribonuclease identified and characterized thus far (1)(2)(3)(4)(5). It has been shown that PARN is an oligomeric, highly processive, and mRNA cap-interacting exonuclease (3, 6 -8).…”

Section: Poly(a)-specific Ribonuclease (Parn)mentioning

confidence: 99%

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

Ren

Martı́nez

Virtanen

2002

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

“…1 is a highly poly(A)-specific 3Ј-exonuclease that efficiently degrades mRNA poly(A) tails (1)(2)(3)(4)(5)(6). PARN is oligomeric, most likely consisting of three subunits (i.e.…”

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confidence: 99%

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

Ren

Kirsebom

Virtanen

2004

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...