A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.     

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode

Poly(A)-specific ribonuclease (PARN) is a processive 3′-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3′ end of the mRNA but also with its 5′ end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m⁷G) cap. The interaction of PARN with the 5′ cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m⁷G triphosphate nucleotide, revealing a novel binding mode for the m⁷G cap. The structure of the m⁷G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m⁷G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two α-helices with an adjacent protein molecule in the crystal lattice.     

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.     

Interaction between a poly(A)-specific ribonuclease and the 5' cap influences mRNA deadenylation rates in vitro

We have used an in vitro system that reproduces in vivo aspects of mRNA turnover to elucidate mechanisms of deadenylation. DAN, the major enzyme responsible for poly(A) tail shortening in vitro, specifically interacts with the 5' cap structure of RNA substrates, and this interaction is greatly stimulated by a poly(A) tail. Several observations suggest that cap-DAN interactions are functionally important for the networking between regulated mRNA stability and translation. First, uncapped RNA substrates are inefficiently deadenylated. Second, a stem-loop structure in the 5' UTR dramatically reduces deadenylation by interfering with cap-DAN interactions. Third, the addition of cap binding protein eIF4E inhibits deadenylation in vitro. These data provide insights into the early steps of substrate recognition that target an mRNA for degradation.     

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.     

Coordination of divalent metal ions in the active site of poly(A)-specific ribonuclease

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 presence of Mg2+. However, in the presence of Mn2+, Zn2+, Co2+, or Cd2+, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions. It was found that the shortest sufficient substrate for PARN activity was adenosine trinucleotide (A3) in the presence of Mg2+ or Cd2+. Interestingly, adenosine dinucleotide (A) was efficiently hydrolyzed in the presence of Mn2+, Zn2+, or Co2+, suggesting that the substrate length requirement for PARN can be modulated by the identity of the divalent metal ion. Finally, introduction of phosphorothioate modifications into the A substrate demonstrated that the scissile bond non-bridging phosphate oxygen in the pro-R position plays an important role during cleavage, most likely by coordinating a catalytically important divalent metal ion. Based on our data we discuss binding and coordination of divalent metal ions in the active site of PARN.     

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.     

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.     

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.     

A nonradioactive assay for poly(a)-specific ribonuclease activity by methylene blue colorimetry

A simple nonradioactive assay, which was based on the specific shift of the absorbance maximum of methylene blue induced by its intercalation into poly(A) molecules, was developed for poly(A)-specific ribonuclease (PARN). A good linear relationship was found between the absorbance at 662 nm and the poly(A) concentration. The assay conditions, including the concentration of methylene blue, the incubation temperature and time, and the poly(A) concentration were evaluated and optimized.     

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.     

The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.     

Combination of Trp and Glu residues for recognition of mRNA cap structure Analysis of mG base recognition site of human cap binding protein (IF-4E) by site-directed mutagenesis

Four mutants of the human cap binding protein (hCBP), in which Trp-102, Glu-103, Asp-104 or Glu-105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants. W102L (Trp-102→Leu) and E105A (Glu-105→Ala), were significantly decreased in comparison with the wild-type hCBP. This result suggest that Trp-102 and Glu-105 are both necessary for the cap binding, and the most probable binding mode with the m⁷G of cap structure is the combination of the stacking by Trp-102 and the hydrogen-bond pairing by Glu-105, as was already proposed from the model studies.     

The RRM1 domain of the poly(A)-binding protein from Saccharomyces cerevisiae is critical to control of mRNA deadenylation

The poly(A)-binding protein PAB1 from the yeast Saccharomyces cerevisiae plays an important role in controlling mRNA deadenylation rates. Deletion of either its RRM1 or proline-rich domain (P domain) severely restricts deadenylation and slows mRNA degradation. Because these large deletions could be having unknown effects on the structure of PAB1, different strategies were used to determine the importance of the RRM1 and P domains to deadenylation. Since the P domain is quite variable in size and sequence among eukaryotes, P domains from two human PABPCs and from Xenopus were substituted for that of PAB1. The resultant PAB1 hybrid proteins, however, displayed limited or no difference in mRNA deadenylation as compared with PAB1. In contrast to the P domain, the RRM1 domain is highly conserved across species, and a systematic mutagenesis of the RRM1 domain was undertaken to identify its functional regions. Several mutations along the RNA-binding surface of RRM1 inhibited deadenylation, whereas one set of mutations on its exterior non-RNA binding surface shifted deadenylation from a slow distributive process to a rapid processive deadenylation. These results suggest that the RRM1 domain is the more critical region of PAB1 for controlling deadenylation and consists of at least two distinguishable functional regions.     

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.     

The RRM domain of human fused in sarcoma protein reveals a non-canonical nucleic acid binding site

Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.     

Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.

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. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(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.     

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.     

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.     

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.