Model of cap‐dependent translation initiation in sea urchin: A step towards the eukaryotic translation regulation network

The large and rapid increase in the rate of protein synthesis following fertilization of the sea urchin egg has long been a paradigm of translational control, an important component of the regulation of gene expression in cells. This translational up‐regulation is linked to physiological changes that occur upon fertilization and is necessary for entry into first cell division cycle. Accumulated knowledge on cap‐dependent initiation of translation makes it suited and timely to start integrating the data into a system view of biological functions. Using a programming environment for system biology coupled with model validation (named Biocham), we have built an integrative model for cap‐dependent initiation of translation. The model is described by abstract rules. It contains 51 reactions involved in 74 molecular complexes. The model proved to be coherent with existing knowledge by using queries based on computational tree logic (CTL) as well as Boolean simulations. The model could simulate the change in translation occurring at fertilization in the sea urchin model. It could also be coupled with an existing model designed for cell‐cycle control. Therefore, the cap‐dependent translation initiation model can be considered a first step towards the eukaryotic translation regulation network. Mol. Reprod. Dev. 77: 257–264, 2010. © 2009 Wiley‐Liss, Inc.     

Lack of in vivo transcription of Acetabularia mediterranea 1175 bp ctDNA fragment homologous to the Drosophila per locus.

The period (per) locus of Drosophila melanogaster has a fundamental role in the expression of biological rhythms. A DNA sequence homologous to a short region of the Drosophila per locus was detected in the chloroplast of Acetabularia mediterranea. A 1175 bp DNA fragment containing the sequence was used as a probe in 'Northern' hybridization experiments. It was found that this DNA was not transcribed or only marginally transcribed in A. mediterranea, at least at the developmental stage just prior to cap formation. It seems that the 1175 bp ctDNA fragment is not involved in the Acetabularia biological rhythm mechanism.     

The effects of concanavalin A on the mobility of lymphocyte surface receptors

It has been found that concanavalin A (Con A) bound to the lymphocyte surface can either induce cap formation or inhibit cap formation of various receptors including those for Con A itself. The expression of these antagonistic activities is highly dependent on the conditions under which cells are incubated with Con A. Incubation with Con A at 37 °C resulted in cap formation in only a small percentage of the cells and inhibited patch and cap formation induced by other reagents such as anti-immunoglobulin. In contrast, incubation of cells with Con A at 4 °C, followed by removal of unbound Con A molecules and elevation of the temperature to 37 °C resulted in cap formation in more than 40 % of the cells. Quantitative analyses suggest that these effects involve cross-linkage of Con A receptors, which occur in two states, mobile and relatively immobile. A model is proposed to explain the various effects of Con A in terms of the association of these receptors with colchicine binding proteins.     

Crystal structure of the interferon-induced ubiquitin-like protein ISG15

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved 3(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba3 activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.     

Influence of electric charge variation at residues 209 and 159 on the interaction of eIF4E with the mRNA 5' terminus

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient protein synthesis in cap-dependent translation. The protein specifically binds the cap structure at the mRNA 5' terminus and facilitates the assembly of the mRNA with other initiation factors and the 40S ribosomal subunit. Phosphorylation of eIF4E is implicated in the regulation of the initiation step of translation. However, the molecular mechanism of this regulation still remains unclear. To address this problem, we have determined the binding affinities of eIF4E specifically mutated at position 209 or 159 for a series of novel mono- and dinucleotide cap analogues by a fluorometric time-synchronized titration method. A 1.5-3-fold reduction in the affinity of cap for the S209E mutant and a 1-2-fold increase in the affinity of cap for the S209K mutant, depending on the negative charge of phosphate chains, indicate that phosphorylation at Ser209 creates electrostatic repulsion between the protein and the negatively charged cap structure. The inhibition of the ability to bind cap analogues by the K159A mutant and its phosphorylated counterpart shows significant participation of Lys159 in the binding of the capped mRNA. Both structural modifications, phosphorylation and the replacement of lysine with alanine, result in an increase in the negative Gibbs free energy of association that is proportional to the length of the cap phosphate chain and additive, i.e., equal to the sum of the individual destabilizing changes of DeltaG degrees. The possible implication of these results for the mechanism of control of eIF4E by phosphorylation, especially for the "clamping model", is discussed.     

Filipin is a reliable in situ marker of ergosterol in the plasma membrane of germinating conidia (spores) of Penicillium discolor and stains intensively at the site of germ tube formation

Filipin, a widely used fluorescent sterol marker is also a potent antibiotic. In this study we address the reliability of filipin as a monitor of ergosterol in fungal cells. A revised staining protocol was developed to minimize any biological effect of the compound. Germinating conidia of Penicillium discolor stained with filipin, displayed a fluorescent cap at the location of germ tube appearance and formation. During germ tube emergence, the fluorescent intensity of the cap increased. This was confirmed by HPLC as an increase of the net cellular ergosterol content. Filipin staining is absent during early germination, while FM dyes, similar molecules, stain the plasma membrane after 1 h. This indicates that the conidial cell wall is no barrier for filipin. To evaluate if filipin does bind ergosterol in situ, natamycin, more specific to ergosterol, was added before filipin staining. This resulted in a marked decrease in fluorescence indicating high ergosterol levels. This was characterized further in ergDelta-mutant cells of Saccharomyces cerevisiae containing altered sterols. Here ergosterol containing cells showed a high fluorescence decrease. Taken together, these data suggest that filipin monitors an ergosterol-enriched cap in germinating conidia at the site of germ tube formation. Furthermore, the sterol-rich cap decreases and reappears after a period of actin disruption. Myriocin that affects sphingolipid synthesis results in an increase of cellular ergosterol and overall filipin fluorescence, but not at the ergosterol cap, where fluorescence is significantly lowered. In conclusion, in this work we have demonstrated an effective revised method for ergosterol staining with filipin and demonstrated its specificity in both Penicillium and Saccharomyces.     

An analysis of morphogenesis of the reproductive whorl of Acetabularia acetabulum

Acetabularia acetabulum (Linn.) P.C. Silva, is a useful system for studying changes in shape because it is large, morphologically complex unicell. The middle, or gametophore lobe of the cap grows radially from the stalk axis as a disc and the fully grown cap can be one of several shapes: flat, concave, convex, and saddle. The shape of the cap normally changes during the first three and a half weeks of reproductive development: individual caps within a population change shape in a stereotypical progression, with the majority proceeding from concave to flat to saddle. Marking the existing surface of caps with carbon grains revealed that the majority of growth occurs near the center, not at the perimeter, of caps. The shape of the mature cap appeared to be independent of algal height, number of gametophores per cap, and final cap diameter. Removing the rhizoid, which contains the nucleus, suggested that the contribution of the nucleus may be important for changes in shape during early cap growth. Based on these data, we present a simple model of cap shape development that suggests both differential growth and biophysical factors may contribute to the final shape of caps of A. acetabulum. Received: 7 January 1998 / Accepted: 7 March 1998     

Insertion of an N7-methylguanine mRNA cap between two coplanar aromatic residues of a cap-binding protein is fast and selective for a positively charged cap

The N7-methylated guanosine (m7G) cap structure, which is found at the 5' ends of mature eukaryotic mRNAs, is critical to a myriad of biological processes. The twenty structures of complexes of cap nucleosides and nucleotides and methylated bases with the vaccinia virus VP39, a cap-specific RNA 2'-O-methyltransferase, which we have determined previously, have revealed the atomic basis of cap binding. The precise insertion and tight fitting of the m7Gua moiety of the cap between two parallel aromatic residues that are spaced only 6.8 A apart governs the high specificity of binding. Here we report the investigation of the reaction mechanism of VP39 with three capped ligands (m7G, m7GpppG, and m7GpppGA3) by fluorescence stopped-flow technique. Cap binding is a simple one-step mechanism with very fast association rate constant (approximately 10(7) M-1 s-1). Moreover, the pH dependence on the association rate constant of m7G binding indicates that only the positively charged keto tautomer of the cap is recognized and bound. The association and dissociation rate constants and affinity constants of the three ligands do not vary greatly, demonstrating that binding is achieved almost entirely by the interactions of m7Gua with two aromatic residues in a cation-pi sandwich.     

Deciphering the mechanistic effects of eIF4E phosphorylation on mRNA‐cap recognition

The mRNA cap‐binding oncoprotein “eIF4E” is phosphorylated at residue S209 by Mnk kinases, and is closely associated with tumor development and progression. Despite being well‐established, mechanistic details at the molecular level of mRNA recognition by eIF4E due to phosphorylation have not been clearly elucidated. We investigated this through molecular modeling and simulations of the S209 phosphorylated derivative of eIF4E and explored the associated implication on the binding of the different variants of mRNA‐cap analogs. A key feature that emerges as a result of eIF4E phosphorylation is a salt‐bridge network between the phosphorylated S209 (pS209) and a specific pair of lysine residues (K159 and K162) within the cap‐binding interface on eIF4E. This interaction linkage stabilizes the otherwise dynamic C‐terminal region of the protein, resulting in the attenuation of the overall plasticity and accessibility of the binding pocket. The pS209‐K159 salt‐bridge also results in an energetically less favorable environment for the bound mRNA‐cap primarily due to electrostatic repulsion between the negative potentials from the phosphates in the cap and those appearing as a result of phosphorylation of S209. These observations collectively imply that the binding of the mRNA‐cap will be adversely affected in the phosphorylated derivative of eIF4E. We propose a mechanistic model highlighting the role of eIF4E phosphorylation as a regulatory tool in modulating eIF4E: mRNA‐cap recognition and its potential impact on translation initiation.     

Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.     

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.     

Insights into the molecular determinants involved in cap recognition by the vaccinia virus D10 decapping enzyme

Decapping enzymes are required for the removal of the 5′-end ^m7GpppN cap of mRNAs to allow their decay in cells. While many cap-binding proteins recognize the cap structure via the stacking of the methylated guanosine ring between two aromatic residues, the precise mechanism of cap recognition by decapping enzymes has yet to be determined. In order to get insights into the interaction of decapping enzymes with the cap structure, we studied the vaccinia virus D10 decapping enzyme as a model to investigate the important features for substrate recognition by the enzyme. We demonstrate that a number of chemically modified purines can competitively inhibit the decapping reaction, highlighting the molecular features of the cap structure that are required for recognition by the enzyme, such as the nature of the moiety at positions 2 and 6 of the guanine base. A 3D structural model of the D10 protein was generated which suggests amino acids implicated in cap binding. Consequently, we expressed 17 mutant proteins with amino acid substitutions in the active site of D10 and found that eight are critical for the decapping activity. These data underscore the functional features involved in the non-canonical cap-recognition by the vaccinia virus D10 decapping enzyme.     

TIMING AND LIGHT REGULATION OF APICAL MORPHOGENESIS DURING REPRODUCTIVE DEVELOPMENT IN WILD-TYPE POPULATIONS OF ACETABULARIA ACETABULUM (CHLOROPHYCEAE)

In the giant unicellular green alga, Acetabularia acetabulum (L.) Silva, development is altered by light. For example, blue light induces the vegetative apex to produce whorls of hairs that encircle the stalk and, later, blue light may trigger reproductive onset. The two goals of this study were to determine when changes in apical shape occur during formation of the reproductive structure, or "cap," and to determine which of these differentiation events require light. The first visible indication of cap initiation was a rounded swelling of the apex, which we call a knob-shaped apex (time = 0 hours). Subsequent changes in shape were a hyaline, knob-shaped apex, reached by 50% of the population 3 h later, and the formation of a whorl of unilobed chambers at 16 h. These chambers became bilobed at 33 h and trilobed at 34 h. Successive sets of cap hairs grew from protuberances found on the surface of the uppermost lobes of the chambers (superior corona). After knob, the remainder of cap formation was largely independent of light. However, the initiation of each set of cap hairs required light. If a recently initiated cap was amputated, the individual recapitulated development, repeating a portion of vegetative morphogenesis (i.e. it made whorls of sterile hairs) before initiating a new cap. The developmental sequence between amputation and initiation of a new cap required light. A model for light-regulated changes in shape at the apex of Acetabularia acetabulum, which integrates whorl and cap formation and encompasses both vegetative and reproductive development of this organism, is presented.     

Asymmetric behavior of severed microtubule ends after ultraviolet- microbeam irradiation of individual microtubules in vitro

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.     

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape.     

New antibiotic resistance Loci in the ribosomal region of yeast mitochondrial DNA

A large number of mitochondrial antibiotic-resistant mutants have been isolated following mutagenesis with manganese. These include several different phenotypic classes of mutants, as distinguished by cross-resistance patterns, that have been found to be allelic at cap1 or ery1; some have been found to be heteroallelic.--Seven chloramphenicol-resistant mutants have been identified that are nonallelic by recombination tests with the three loci (cap1, spi1 and ery1) previously identified in the ribosomal region. Four of these are allelic with each other and define a new locus, cap3; two others are allelic and define another new locus, cap2; the seventh maps at yet a different locus, cap4. One new spiramycin-resistant mutant has been identified that defines still another new locus, spi2. A variety of genetic techniques have been used to map these loci within the ribosomal region of the mitochondrial genome.-Manganese has been shown to be effective in inducing the mutation from omega(-) to omega(n) in many mutants that experience a simultaneous mutation at the closely linked cap1 locus. The omega(n) mutation has also been described in the cap4 mutant, and this locus has been shown to be more closely linked to omega than cap1 is to omega.     

Myoglobin models and steric origins of the discrimination between O2 and CO

 Synthetic models of the myoglobin active site have provided much insight into factors that affect CO and O₂ binding in the proteins. "Capped" and "pocket" metal porphyrin systems have been developed to probe how steric factors affect ligand binding and ultimately to elucidate important aspects of the mechanism of CO discrimination in the proteins. These model porphyrins are among the most thoroughly characterized systems to date. From the twenty-one known crystal structures, analysis of the types of distortion that occur upon ligand binding under the cap, including porphyrin doming and ruffling, lateral and horizontal movement of the cap, and bending and tilting of the Fe–C–O bond, provides an indication of how steric interactions will affect structure in Hb and Mb. The model porphyrin systems discussed range from those that discriminate against O₂ binding compared to biological systems to those with similar CO and O₂ binding strength to myoglobin, and also to those that bind both O₂ and CO very weakly or not at all. The primary type of distortion observed upon CO binding is vertical or lateral movement of the cap and some ruffling of the porphyrin plane. Minimal bending or tilting of the M–C–O bond is observed, suggesting that the Fe–C–O bending that has been found from crystal structures of the hemoproteins is unlikely. Received, accepted: 23 May 1997     

Purification and characterization of the m7G(5')pppN-pyrophosphatase from human placenta

A purification scheme has been developed for the m7G(5')pppN-pyrophosphatase from human placenta. The 1400-fold purified placental enzyme exhibited physical and enzymatic properties similar to those previously reported for a crude preparation of the human m7G(5')pppN-pyrophosphatase obtained from HeLa cells. Polyacrylamide gel analysis of enzyme fractions at different stages of purification revealed a Mr = 40,000 polypeptide that increased in relative concentration as the specific activity of the enzyme fractions increased. Copurification of this polypeptide with m7G(5')pppN-pyrophosphatase activity suggests the possibility that the 81,000-dalton native enzyme is a dimer composed of subunits of identical molecular weight. The highly purified placental enzyme, like the crude HeLa enzyme, failed to hydrolyze the cap moiety of intact mRNA even under conditions known to reduce mRNA secondary structure. Moreover, when a series of capped oligonucleotides that differed progressively in chain length by a factor of one nucleotide was tested as substrate, the rate of enzyme-catalyzed cap hydrolysis decreased as the chain length increased. The purified placental enzyme failed to release m7pG from oligonucleotides containing the cap and 3 or more additional nucleotides. These results are discussed in terms of the probable biological function of the m7G(5')pppN-pyrophosphatase.