The mRNA cap structure stimulates rate of poly(A) removal and amplifies processivity of degradation

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive, and cap-interacting 3' exonuclease. We have studied how the m7G(5')ppp(5')G cap structure affects the activity of PARN. It is shown that the cap has four distinct effects: (i) It stimulates the rate of deadenylation if provided in cis; (ii) it inhibits deadenylation if provided at high concentration in trans; (iii) it stimulates deadenylation if provided at low concentration in trans; and (iv) it increases the processivity of PARN when provided in cis. It is shown that the catalytic and cap binding sites on PARN are separate. The important roles of the 7-methyl group and the inverted guanosine residue of the cap are demonstrated. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. Complex formation does not require a cap structure on the RNA substrate. The multiple effects of cap are all accounted for by a simple, kinetic model that takes the processivity of PARN into account.     

Cap-dependent deadenylation of mRNA

Poly(A) tail removal is often the initial and rate-limiting step in mRNA decay and is also responsible for translational silencing of maternal mRNAs during oocyte maturation and early development. Here we report that deadenylation in HeLa cell extracts and by a purified mammalian poly(A)-specific exoribonuclease, PARN (previously designated deadenylating nuclease, DAN), is stimulated by the presence of an m(7)-guanosine cap on substrate RNAs. Known cap-binding proteins, such as eIF4E and the nuclear cap-binding complex, are not detectable in the enzyme preparation, and PARN itself binds to m(7)GTP-Sepharose and is eluted specifically with the cap analog m(7)GTP. Xenopus PARN is known to catalyze mRNA deadenylation during oocyte maturation. The enzyme is depleted from oocyte extract with m(7)GTP-Sepharose, can be photocross-linked to the m(7)GpppG cap and deadenylates m(7)GpppG-capped RNAs more efficiently than ApppG-capped RNAs both in vitro and in vivo. These data provide additional evidence that PARN is responsible for deadenylation during oocyte maturation and suggest that interactions between 5' cap and 3' poly(A) tail may integrate translational efficiency with mRNA stability.     

Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC)

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.     

A unique system for regulating mitochondrial mRNA poly(A) status and stability in plants

Poly(A) status is the major determinant of mRNA stability, even in endosymbiotic organelles. Poly(A) specific ribonuclease (PARN) is distributed widely among eukaryotes and has been shown to regulate the poly(A) status of cytoplasmic mRNA in various organisms. Surprisingly, our recent study revealed that PARN also directly regulates poly(A) status of mitochondrial mRNA in Arabidopsis. In this addendum, we discuss whether this mitochondrial function of PARN is common in plants and why PARN has been assigned such a unique function.     

Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU

The AU-rich element (ARE) in the 3' untranslated region of unstable mRNAs mediate their rapid degradation. ARE binding proteins (AUBPs) have been described that either stabilize or otherwise degrade ARE-mRNAs by recruiting the exosome, a complex of 3'-to-5' exoribonucleases. We have identified RHAU, a putative DExH RNA helicase that was isolated in association with the ARE of urokinase plasminogen activator mRNA (ARE(uPA)). RHAU physically interacts with the deadenylase PARN and the human exosome and enhances the deadenylation and decay of ARE(uPA)-mRNAs. An alternatively spliced isoform of RHAU that localized to the cytoplasm had a more pronounced effect on ARE(uPA)-mRNA destabilization than full-length RHAU. Furthermore, the ATPase activity of RHAU is essential for its mRNA-destabilizing function. ARE(uPA)-mRNA recognition by RHAU may be mediated through its RNA-dependent interaction with the AUBPs HuR and NFAR1. A model is presented to describe the action of RHAU in ARE(uPA)-directed mRNA turnover.     

CUG-BP binds to RNA substrates and recruits PARN deadenylase

CUG-BP is the human homolog of the Xenopus EDEN-BP, which was shown previously to bind to mRNAs, such as c-mos, that exhibit rapid deadenylation following fertilization of the oocyte. While several studies have focused on roles of CUG-BP as a splicing or translation regulator in mammalian cells, its role in mRNA decay has not been examined in detail. Here, we have used an in vitro deadenylation assay to dissect the function of CUG-BP in the decay of two ARE-containing mRNAs: c-fos and TNFalpha. CUG-BP binds specifically to both of these RNAs and stimulates poly(A) shortening by PARN. Moreover, CUG-BP interacts with PARN in extracts by coimmunoprecipitation, and this interaction can be recapitulated using recombinant proteins. CUG-BP, therefore, is the first RNA-binding protein shown to directly recruit a deadenylase to an RNA substrate.     

Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3'-->5' exonuclease specific for adenylate residues, generates 5'-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.     

PARN deadenylase is involved in miRNA-dependent degradation of TP53 mRNA in mammalian cells

mRNA deadenylation is under the control of cis-acting regulatory elements, which include AU-rich elements (AREs) and microRNA (miRNA) targeting sites, within the 3′ untranslated region (3′ UTRs) of eukaryotic mRNAs. Deadenylases promote miRNA-induced mRNA decay through their interaction with miRNA-induced silencing complex (miRISC). However, the role of poly(A) specific ribonuclease (PARN) deadenylase in miRNA-dependent mRNA degradation has not been elucidated. Here, we present evidence that not only ARE- but also miRNA-mediated pathways are involved in PARN-mediated regulation of the steady state levels of TP53 mRNA, which encodes the tumor suppressor p53. Supporting this, Argonaute-2 (Ago-2), the core component of miRISC, can coexist in complexes with PARN resulting in the activation of its deadenylase activity. PARN regulates TP53 mRNA stability through not only an ARE but also an adjacent miR-504/miR-125b-targeting site in the 3′ UTR. More importantly, we found that miR-125b-loaded miRISC contributes to the specific recruitment of PARN to TP53 mRNA, and that can be reverted by the ARE-binding protein HuR. Together, our studies provide new insights into the role of PARN in miRNA-dependent control of mRNA decay and into the mechanisms behind the regulation of p53 expression.     

Analysis of ABA hypersensitive germination2 revealed the pivotal functions of PARN in stress response in Arabidopsis

Accumulating evidence suggests that mRNA degradation systems are crucial for various biological processes in eukaryotes. Here we provide evidence that an mRNA degradation system is associated with some plant hormones and stress responses in plants. We analysed a novel Arabidopsis abscisic acid (ABA)-hypersensitive mutant, ahg2-1, that showed ABA hypersensitivity not only in germination, but also at later developmental stages, and that displayed pleiotropic phenotypes. We found that ahg2-1 accumulated more endogenous ABA in seeds and mannitol-treated plants than did the wild type. Microarray experiments showed that the expressions of ABA-, salicylic acid- and stress-inducible genes were increased in normally grown ahg2-1 plants, suggesting that the ahg2-1 mutation somehow affects various stress responses as well as ABA responses. Map-based cloning of AHG2 revealed that this gene encodes a poly(A)-specific ribonuclease (AtPARN) that is presumed to function in mRNA degradation. Detailed analysis of the ahg2-1 mutation suggests that the mutation reduces AtPARN production. Interestingly, expression of AtPARN was induced by treatment with ABA, high salinity and osmotic stress. These results suggest that both upregulation and downregulation of gene expression by the mRNA-destabilizing activity of AtPARN are crucial for proper ABA, salicylic acid and stress responses.     

聚腺苷酸特异性核糖核酸酶(PARN)的功能多样性

聚腺苷酸尾的降解对于mRNA的质量控制和转录后基因调控十分重要. 在真核生物中,去腺苷酸化是mRNA降解和翻译沉默的首要限速步骤. 3′核糖核酸外切酶--聚腺苷酸特异性核糖核酸酶（poly(A)-specific ribonuclease,PARN）能够高效降解真核生物mRNA的聚腺苷酸尾. PARN不仅在降解mRNA poly(A)尾中发挥关键的作用,还参与DNA损伤、非编码RNA的加工成熟以及肿瘤等疾病过程. PARN是一种多功能酶分子,本文就PARN发现、结构、催化机制和功能多样性进行综述.     

Rapid deadenylation and Poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos

The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.     

Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4

Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. Here we identify a novel level of nanos translational control. We show that the Smaug protein bound to the nanos 3' UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. We further propose a mechanism for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. We show that Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development.     

Effect of magnesium ions on the thermal stability of human poly(A)-specific ribonuclease

Poly(A)-specific ribonuclease (PARN), a member of the DEDD family, is a key enzyme involved in the deadenylation of mRNA in higher eukaryotic cells. In this research, it was found that Mg(2+) could protect PARN against thermal inactivation by increasing the midpoint of inactivation and decreasing the inactivation rate. This protective effect was unique to Mg(2+) in a concentration-dependent manner. However, the thermal unfolding and aggregation was promoted by the addition of Mg(2+) at high temperatures. These results revealed that Mg(2+) might have dual effects on PARN stability: protecting the active site but endangering the overall structural stability.     

Localization of AU-rich element-containing mRNA in cytoplasmic granules containing exosome subunits

Eukaryotic mRNAs can be degraded in either decapping/5'-to-3' or 3'-to-5' direction after deadenylation. In yeast and mammalian cells, decay factors involved in the 5'-to-3' decay pathway are concentrated in cytoplasmic processing bodies (P bodies). The mechanistic steps and localization of mammalian mRNA decay are still not completely understood. Here, we investigate functions of human mRNA decay enzymes in AU-rich element (ARE)-mediated mRNA decay (AMD) and find that the deadenylase, poly(A) ribonuclease PARN, and enzymes involved in the 5'-to-3' and 3'-to-5' decay pathways are required for AMD. The ARE-containing reporter mRNA accumulates in discrete cytoplasmic granular structures, which are distinct from P bodies and stress granules. These granules consist of poly(A)-specific ribonuclease, exosome subunits, and decay-promoting ARE-binding proteins. Inhibition of AMD increases accumulation of ARE-mRNA in these granules. We refer to these structures as cytoplasmic exosome granules and suggest that some AMD may occur in these granules.