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.     

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability.     

Structural Model of the Human BTG2–PABPC1 Complex by Combining Mutagenesis,NMR Chemical Shift Perturbation Data and Molecular Docking

Degradation of cytoplasmic mRNA in eukaryotes involves the shortening and removal of the mRNA poly(A) tail by poly(A)-selective ribonuclease (deadenylase) enzymes. In human cells, BTG2 can stimulate deadenylation of poly(A) bound by cytoplasmic poly(A)-binding protein PABPC1. This involves the concurrent binding by BTG2 of PABPC1 and the Caf1/CNOT7 nuclease subunit of the Ccr4-Not deadenylase complex. To understand in molecular detail how PABPC1 and BTG2 interact, we set out to identify amino acid residues of PABPC1 and BTG2 contributing to the interaction. To this end, we first used algorithms to predict PABPC1 interaction surfaces. Comparison of the predicted interaction surface with known residues involved in the binding to poly(A) resulted in the identification of a putative interaction surface for BTG2. Subsequently, we used pulldown assays to confirm the requirement of PABPC1 residues for the interaction with BTG2. Analysis of RNA-binding by PABPC1 variants indicated that PABPC1 residues required for interaction with BTG2 do not interfere with poly(A) binding. After further defining residues of BTG2 that are required for the interaction with PABPC1, we used information from published NMR chemical shift perturbation experiments to guide docking and generate a structural model of the BTG2-PABPC1 complex. A quaternary poly(A)-PABPC1-BTG2-Caf1/CNOT7 model showed that the 3′ end of poly(A) RNA is directed towards the catalytic centre of Caf1/CNOT7, thereby providing a rationale for enhanced deadenylation by Caf1/CNOT7 in the presence of BTG2 and PABPC1.     

Temporal Control of Metabolic Amplitude by Nocturnin

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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.     

一个独特的RNA酶A抗性复合物在腺病毒L1 poly(A)位点的上游形成

在腺病毒交替的ｐｏｌｙ（Ａ）位点使用过程中,靠近主要晚期启动子的Ｌ１ ｐｏｌｙ（Ａ）位点起着主导的作用。前期的实验已经发现,在Ｌ１ ｐｏｌｙ（Ａ）位点的上游存在一个ＲＮＡ的抑制元件叶ＵＲＥ,缺失ＵＲＥ可以使模拟小基因的ｐｏｌｙ（Ａ）进入病毒晚期的感染方式。现将Ｌ１ ｐｏｌｙ（Ａ）位点单独游离出来,用体外的紫外交联的方法对其进行研究,结果发现在没有紫外光照射的情况下,仍有一组小于３０ｋＤ独特的ＲＮＡ     

Messenger RNA Turnover in Eukaryotes: Pathways and Enzymes

The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5′ exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3′ exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.     

A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila

The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.     

Effects of 3′deoxyadenosine and actinomycin D on RNA synthesis in toad bladder: Analysis of response to aldosterone

Summary Previous studies have shown that aldosterone increases transepithelial active Na⁺ transport after a latent period of about 60 min and incorporation of³H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toadBufo marinus. 3deoxyadenosine (30 g/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na⁺ transport measured as short-circuit current (scc). The incorporation of³H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 g/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of³H-uridine into poly(A)(+)RNA by 68 to 75%. 3deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dependent Na⁺ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA; however, 3deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA.     

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.