The mechanism and regulation of deadenylation: Identification and characterization of Xenopus PARN

108

118

The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae. Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex. Here, we present evidence that mCAF1 is a processive, 3 -5 -RNase with a preference for poly(A) substrates. Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme. In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates. The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly(A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3 -untranslated region sequences. The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1. Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1. These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function.

Section: Table Imentioning

confidence: 99%

Mouse CAF1 Can Function As a Processive Deadenylase/3′–5′-Exonuclease in Vitro but in Yeast the Deadenylase Function of CAF1 Is Not Required for mRNA Poly(A) Removal

Viswanathan

Ohn

Chiang

et al. 2004

108

118

“…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).…”

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

confidence: 99%

“…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).…”

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.…”

mentioning

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...