Structural basis of m7GpppG binding to the nuclear cap-binding protein complex

The 7-methyl guanosine cap structure of RNA is essential for key aspects of RNA processing, including pre-mRNA splicing, 3' end formation, U snRNA transport, nonsense-mediated decay and translation. Two cap-binding proteins mediate these effects: cytosolic eIF-4E and nuclear cap-binding protein complex (CBC). The latter consists of a CBP20 subunit, which binds the cap, and a CBP80 subunit, which ensures high-affinity cap binding. Here we report the 2.1 A resolution structure of human CBC with the cap analog m7GpppG, as well as the structure of unliganded CBC. Comparisons between these structures indicate that the cap induces substantial conformational changes within the N-terminal loop of CBP20, enabling Tyr 20 to join Tyr 43 in pi-pi stacking interactions with the methylated guanosine base. CBP80 stabilizes the movement of the N-terminal loop of CBP20 and locks the CBC into a high affinity cap-binding state. The structure for the CBC bound to m7GpppG highlights interesting similarities and differences between CBC and eIF-4E, and provides insights into the regulatory mechanisms used by growth factors and other extracellular stimuli to influence the cap-binding state of the CBC.     

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.     

Translation mediated by the nuclear cap-binding complex is confined to the perinuclear region via a CTIF–DDX19B interaction

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5′-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5′-cap of a newly exported mRNA. The resulting CBP80–CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.     

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.     

Cdc42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex

Cdc42 is a low molecular weight GTP-binding protein that plays a key regulatory role in a variety of cellular activities. The importance of the coordination of different cell functions by Cdc42 is underscored by the fact that a constitutively active Cdc42 mutant induces cellular transformation. In this study, we describe a novel function for Cdc42: its ability to stimulate pre-messenger RNA splicing. This activity is dependent on cysteine 37 in the effector loop of Cdc42 but is not dependent on cell growth. A likely candidate protein for mediating the Cdc42 effects on pre-mRNA splicing is the nuclear RNA cap-binding complex (CBC), which plays a key role in an early step of cap-dependent RNA splicing. Activation of the CBC by Cdc42 can be inhibited by rapamycin. Additionally, phosphatidylinositol 3-kinase and the Cdc42 effector, pp70 S6 kinase, stimulate the RNA cap-binding activity of the CBC. S6 kinase may directly target the CBC in vivo as it can phosphorylate the 80-kDa subunit of the CBC, CBP80, at residues that are subject to a growth factor-dependent and rapamycin-sensitive phosphorylation in vivo. Together these data suggest the involvement of a Cdc42-S6 kinase pathway in the regulation of RNA splicing, mediated by an increase in capped RNA binding by the CBC, as well as raise the possibility that the effects of Cdc42 on cell growth may be due in part to its regulation of RNA processing.     

NOVEL WAY OF CAPPING mRNA TRIMER AND STUDIES OF ITS INTERACTION WITH HUMAN NUCLEAR CAP-BINDING COMPLEX

Binding of mRNA 5′ cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.     

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.     

Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex

Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.     

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.     

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.     

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.     

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.     

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.