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

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg(2 + ) which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.     

Poly(A)-specific ribonuclease (PARN): An allosterically regulated,processive and mRNA cap-interacting deadenylase

Abstract

Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m⁷G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3′–5′ exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.     

Kinetic and in silico analysis of the slow-binding inhibition of human poly(A)-specific ribonuclease (PARN) by novel nucleoside analogues

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that efficiently degrades poly(A) tails and regulates, in part, mRNA turnover rates. We have previously reported that adenosine- and cytosine-based glucopyranosyl nucleoside analogues with adequate tumour-inhibitory effect could effectively inhibit PARN. In the present study we dissect the mechanism of a more drastic inhibition of PARN by novel glucopyranosyl analogues bearing uracil, 5-fluorouracil or thymine as the base moiety. Kinetic analysis showed that three of the compounds are competitive inhibitors of PARN with K_i values in the low μM concentration and significantly lower (11- to 33-fold) compared to our previous studies. Detailed kinetic analysis of the most effective inhibitor, the uracil-based nucleoside analogue (named U1), revealed slow-binding behaviour. Subsequent molecular docking experiments showed that all the compounds which inhibited PARN can efficiently bind into the active site of the enzyme through specific interactions. The present study dissects the inhibitory mechanism of this novel uracil-based compound, which prolongs its inhibitory effect through a slow-binding and slow-release mode at the active site of PARN, thus contributing to a more efficient inhibition. Such analogues could be used as leading compounds for further rationale design and synthesis of efficient and specific therapeutic agents. Moreover, our data reinforce the notion that human PARN can be established as a novel molecular target of potential anti-cancer agents through lowering mRNA turnover rates.     

Allosteric regulation of human poly(A)-specific ribonuclease by cap and potassium ions

Poly(A)-specific ribonuclease (PARN), a multi-domain dimeric enzyme, is a deadenylase in higher vertebrates and plants with the unique property of cap-dependent catalysis and processivity. We found that PARN is an allosteric enzyme, and potassium ions and the cap analogue were effectors with binding sites located at the RRM domain. The binding of K⁺ to the entire RRM domain led to an increase of substrate-binding affinity but a decrease in the cooperativity of the substrate-binding site, while the binding of the cap analogue decreased both the catalytic efficiency and the substrate-binding affinity. The dissimilar kinetic properties of the enzymes with and without the entire RRM domain suggested that the RRM domain played a central role in the allosteric communications of PARN regulation. The allostery is proposed to be important to the multi-level regulation of PARN to achieve precise control of the mRNA poly(A) tail length.     

Role of the RRM domain in the activity, structure and stability of poly(A)-specific ribonuclease

Poly(A) specific ribonuclease (PARN), which contains a catalytic domain and two RNA-binding domains (R3H and RRM), acts as a key enzyme in eukaryotic organisms to regulate the stability of mRNA by degrading the 3' poly-(A) tail. In this research, the activity, structure and stability were compared between the full-length 74kDa PARN, the proteolytic 54kDa fragment with half of the RRM, and a truncated 46kDa form completely missing the RRM. The results indicated that the 46kDa one had the lowest activity and substrate binding affinity, the most hydrophobic exposure in the native state and the least stability upon denaturation. The dissimilarity in the activity, structure and stability of the three PARNs revealed that the entire RRM domain not only contributed to the substrate binding and efficient catalysis of PARN, but also stabilized the overall structures of the protein. Spectroscopic experiments suggested that the RRM domain might be structurally adjacent to the R3H domain, and thus provide a basis for the cooperative binding of poly(A) by the two RNA-binding domains as well as the catalytic domain.     

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K⁺ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K⁺, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.     

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.     

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.     

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.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

Expression and purification of recombinant poly(A)-specific ribonuclease (PARN)

PARN is a poly(A)-specific ribonuclease that degrades the poly(A) tail of mRNA. We have established conditions for expressing soluble recombinant human PARN. We investigated different Escherichia coli strains, expression vectors, media and growth conditions. We found that PARN expressed from pET33 in BL21(DE3) grown in TB and induced at OD595 approximately 1 with 1 mM IPTG yielded mg amounts of soluble PARN per litre culture. Further, a purification protocol was established to purify PARN. We use His-tag affinity chromatography, HiTrap Q HP ion exchange chromatography and 7-Me-GTP-Sepharose affinity chromatography. This purification procedure render a 90-95% pure PARN. Purified recombinant PARN has enzymatic activity and will be used for further mechanistic and structural studies.     

Self-association of poly(A)-specific ribonuclease (PARN) triggered by the R3H domain

Poly(A)-specific ribonuclease (PARN) is a deadenylase with three RNA-binding domains (the nuclease, R3H and RRM domains) and a C-terminal domain. PARN participates in diverse physiological processes by regulating mRNA fates through deadenylation. PARN mainly exists as a dimer in dilute solutions. In this research, we found that PARN could self-associate into tetramer and high-order oligomers both in vitro and in living cells. Mutational and spectroscopic analysis indicated that PARN oligomerization was triggered by the R3H domain, which led to the solvent-exposed Trp219 fluorophore to become buried in a solvent-inaccessible microenvironment. The RRM and C-terminal domains also played a role in modulating the dissociation rate of the tetrameric PARN. Enzymatic analysis indicated that tetramerization did not affect the catalytic behavior of the full-length PARN and truncated enzymes containing the RRM domain, which might be caused by the high propensity of the dimeric proteins to self-associate into oligomers. Tetramerization significantly enhanced the catalytic activity and processivity of the truncated form with the removal of the RRM and C-terminal domains. The results herein suggested that self-association might be one of the regulation methods for PARN to achieve a highly regulated deadenylase activity. We propose that self-association may facilitate PARN to concentrate around the target mRNAs by restricted diffusion.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Distinct roles of the R3H and RRM domains in poly(A)-specific ribonuclease structural integrity and catalysis

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3′- to 5′-end direction with the release of 5′-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.     

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.     

Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.