mRNA Cap Structure Research Articles

Cleavage of the 5‘ Cap Structure of mRNA by a Europium(III) Macrocyclic Complex with Pendant Alcohol Groups

Cellular and viral RNAs, synthesized by RNA polymerase II, have a unique chemical structure at their 5‘ termini, 5‘ m7GpppNNN... 3‘, referred to as the 5‘ cap. This structure plays a pivotal role i...

Viral and Cellular Enzymes Involved in Synthesis of mRNA Cap Structure

Viral and Cellular Enzymes Involved in Synthesis of mRNA Cap Structure

EIF4G Dramatically Enhances the Binding of eIF4E to the mRNA 5′-Cap Structure

The cap structure, m7GpppN, is present at the 5'-end of all eukaryotic cellular (except organellar) mRNAs. Initiation of translation is mediated by the multisubunit initiation factor eIF4F, which binds the cap structure via its eIF4E subunit and facilitates the binding of mRNA to ribosomes. Here, we used recombinant proteins to reconstitute the cap recognition activity of eIF4F in vitro. We demonstrate that the interaction of eIF4E with the mRNA 5'-cap structure is dramatically enhanced by eIF4G, as determined by a UV-induced cross-linking assay. Furthermore, assembly of the eIF4F complex at the cap structure, as well as ATP hydrolysis, is shown to be a requisite for the cross-linking of another initiation factor, eIF4B, to the cap structure. In addition, the stimulatory effect of eIF4G on the cap recognition of eIF4E is inhibited by the translational repressor, 4E-BP1. These results suggest that eIF4E initially interacts with the mRNA cap structure as part of the eIF4F complex.

Differential effect of modified capped RNA substrates on influenza virus transcription

The RNA-dependent RNA polymerase of influenza virus transcribes messenger RNA through a unique cap scavenging mechanism. Viral enzyme binds to the cap structure of host mRNA, cleaves the molecule 9–15 bases downstream of the cap, and uses the short capped oligonucleotide as a primer for mRNA synthesis. Previously, we have shown that the viral polymerase can efficiently bind capped RNAs shorter than 9 nucleotides in length, but the viral enzyme can not utilize these RNAs as primers. For this reason, these short capped oligonucleotides are potent inhibitors of influenza virus transcription. In these studies, it is now shown that short capped oligomers inhibit capped-RNA dependent transcription at the initial step of cap binding. In contrast, low concentrations of these short capped RNAs can actually stimulate viral transcription primed with high concentrations of the dinucleotide ApG. Another capped RNA derivative containing phosphorothioate oligonucleotides was also investigated as a potential polymerase inhibitor. This longer capped RNA was able to bind to the polymerase, but could not be cleaved to primer length by the enzyme associated endonuclease. Thus, the capped phosphorothioate RNA inhibited cap-primed transcription at the step of cap binding. However, in contrast to the short capped oligonucleotide, it also inhibited ApG primed viral transcription.

High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 × 107/10 μg starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

Mutation of the Cysteine Residues in Human Initiation Factor 4E: Effects on mRNA Cap Binding Ability

To investigate the role of four Cys residues of eukaryotic initiation factor (eIF)-4E in the recognition of the mRNA cap structure, single, double and quadruple mutant genes which encoded the Ala residues in the place of the respective Cys residues were prepared using a synthetic human eIF-4E gene by the site-directed mutagenesis, and were expressed inE. coliwith the same way as the wild type. The cap binding abilities of respective mutated eIF-4Es were compared with that of the wild-type by m7GTP affinity chromatography. The results suggest that, although all four of Cys residues participate in the recognition of the mRNA cap structure, they contribute indirectly to the stabilization of overall tertiary structure, especially of the cap binding pocket, rather than by direct interaction. A comparison among the cap binding abilities of single, double and quadruple mutants indicated no existence of internal disulfide bonds in eIF-4E.

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

Intracellular targeting and mRNA interactions of the eukaryotic translation initiation factor eIF4E in the yeast Saccharomyces cerevisiae

The 5′ cap structure of eukaryotic mRNAs is believed to play a role in a number of cellular processes, including pre-mRNA splicing, nuclear export and translation. An essential cap-binding protein that is likely to mediate the participation of the cap in at least one of these processes is the eukaryotic translation initiation factor eIF4E. This protein is thought to facilitate the initial ribosomal interaction with the 5′ end of the mRNA, involving the binding of eIF4E to the cap in the cytoplasm. Yet the subcellular distribution and mechanism of targeting of eIF4E has been an unresolved issue. We have therefore examined whether eIF4E in the yeast Saccharomyces cerevisiae is directed to the nucleus by virtue of a nuclear localization sequence (NLS) in its amino acid sequence. eIF4E was fused with the ‘marker proteins’ yeast invertase and jellyfish green fluorescent protein. The distribution of these fusions could be followed using immunofluorescence and confocal microscopy of protoplasts and whole cells. These and other fusions were used to show that while yeast eIF4E does not possess an efficiently functioning NLS, it can be transported into the nucleus if provided with a known active NLS. However, an NLS-eIF4E fusion of this type cannot be stably supported by the cell, most likely because of its inhibitory effects when present in large quantities in the nucleus, whereas an NLS fusion with a mutant form of eIF4E that has reduced cap-affinity is tolerated.

Analysis of the mRNA Cap-Binding Ability of Human Eukaryotic Initiation Factor-4E by Use of Recombinant Wild-Type and Mutant Forms

In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5'-triphosphate-Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF-4E genes, which were constructed by site-directed mutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues. Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket.

Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

MRNA decapping activities and their biological roles

The 5′ cap structure of eukaryotic mRNAs is significant for a variety of cellular events and also serves to protect mRNAs from premature degradation. Analysis of mRNA decay in Saccharomyces cerevisiae has shown that removal of the 5′ cap structure is a key step in the turnover of many yeast mRNAs, and that this decapping is carried out by Dcplp. In addition to the yeast decapping enzyme, other activities that can cleave the 5′ cap structure have been described. These include two mammalian enzymes and two viral activities that cleave cellular mRNA cap structures as part of their life cycle. Here we review these various decapping activities and discuss their biological roles.

A facile synthesis of 2-azidoadenosine derivatives from guanosine as photoaffinity probes

2-Azidoadenosine ( 1) was synthesized in an overall yield of 49% from guanosine via the reaction of 9-(2′,3′,5′-tri- O-acetyl- β-D-ribofuranosyl)-2-amino-6-chloropurine ( 2) with isoamyl nitrite and trimethylsilyl azide under neutral and anhydrous conditions. As photoaffinity probes, ATP analogues and the cap structure of eukaryotic mRNA bearing 2-azidoadenosine were synthesized.

Design, synthesis, and analysis of antisense oligonucleotide metal complex conjugates which react upon the 5' cap structure of mRNA

Design, synthesis, and analysis of antisense oligonucleotide metal complex conjugates which react upon the 5' cap structure of mRNA

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.

Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a broad spectrum of transformed cell lines.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Over-expression of eIF-4E in fibroblasts results in their malignant transformation. However, no information on eIF-4E expression in established transformed cell lines has been available. We report here that a variety of tumor cell lines, chemically, virally and oncogenically transformed, exhibit elevated levels of eIF-4E mRNA expression as compared to their normal counterparts. Overexpression of eIF-4E, which is normally rate-limiting in protein synthesis, may stimulate the translation of regulatory and oncogenic proteins involved in transformation.

Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation

Hormone-induced meiotic maturation of the Xenopus oocyte is regulated by complex changes in protein phosphorylation. It is accompanied by a stimulation in the rate of translation, manifest at the level of polypeptide chain initiation. At laser times in the maturation process, this reflects an increased ability for mRNA to interact with the 40 S ribosomal subunit. In mammalian cells there is growing evidence for the regulation of translation by phosphorylation of ribosomal protein S6 and of initiation factors responsible for the binding of mRNA to ribosomes. In this report, we show that although the 70 kDa form of S6 kinase is activated within 1.5 hours in response to progesterone or insulin, a time critical for protein synthesis, its activation is not required for hormone-induced stimulation of translation rates or maturation. In response to progesterone, activation of translation occurs in parallel with enhanced phosphate labelling of eIF-4 alpha and eIF-4 gamma and eIF-4F complex formation, events which are thought to facilitate the interaction of eIF-4F with the mRNA cap structure. However, with insulin, activation of translation occurs prior to detectable de novo phosphorylation of eIF-4F, although a small enhancement of turnover of phosphate on eIF-4 alpha may occur at this early time. With either hormone, enhanced phosphate labelling of eIF-4 alpha is shown to reflect activation of eIF-4 alpha kinase(s), which coincides temporally with activation of p42 MAP and p90rsk kinases. The possible role of initiation factor modification on increased translation rates during meiotic maturation is discussed.

The proto‐oncogene/translation factor eIF4E: A survey of its expression in breast carcinomas

The eukaryotic translation initiation factor eIF-4E binds to the cap structure of mRNAs as one component of the eIF-4 translation initiation complex, which mediates the recruitment of mRNA to the ribosomes. Overexpression of eIF-4E can result in oncogenic transformation and uncontrolled growth of mammalian cells, presumably by facilitating the expression of growth-control gene products which are normally translationally repressed. Whereas the mechanism of eIF-4E-mediated transformation is being actively pursued, clinical investigations into the expression of eIF-4E in prevalent human cancers are lacking. We have recently initiated a screen of breast carcinomas by probing with eIF-4E antiserum. Using Western blots, we have analyzed the level of eIF-4E in 38 carcinomas, 7 normal samples and 3 fibroadenomas. We found that eIF-4E was elevated 3- to 10-fold in virtually all the carcinomas we analyzed, but not in fibroadenomas. This analysis was also confirmed by immunohistological staining in situ, showing that overexpression of eIF-4E can be readily identified at the single-cell level. Our results suggest that an elevation of eIF-4E may be an essential component in the development of breast cancer.

Gene expression of human eukaryotic initiation factor-4E for protein synthesis and study of its recognition mechanism of mRNA cap structure

Being stimulated by the insights from model studies that (i) the intimate combination of hydrogen-bonding pairing and aromatic stacking interactions is important for the specific binding of guanine base by peptide and (ii) the pi-pi stacking force of Trp is significantly strengthened by the guanine N7-methylation (m7G), this research project was started, because (a) the mRNA cap structure is characterized by the existence of m7G and (b) an eukaryotic initiation factor-4E (eIF-4E), a protein which specifically recognizes the mRNA cap structure and opens the protein biosynthesis, contains 8 Trp residues irrespective of its relatively low molecular weight of about 25 kDa. In order to prepare the sufficient amount of sample for carrying out the analysis of the recognition mechanism of mRNA cap structure by eIF-4E at the atomic level, firstly, the expression of human eIF-4E gene in Escherichia coli was attempted. An artificial gene encoding for human eIF-4E was chemically synthesized and succeeded in the expression with two different forms, i.e., as a fusion protein with human growth hormone and a direct expression of soluble protein. The isolation of eIF-4E and its purification procedure using the m7GTP affinity chromatography were accomplished. It was shown by spectroscopic methods that the recombinant eIF-4E exhibits essentially the same tertiary structure as the native one and the binding ability with mRNA cap analog was identical with each other. In order to analyze the functional amino acid residues which are essential for specific recognition of mRNA cap structure, next a series of eIF-4E mutants were prepared by the site-directed mutagenesis, and His37, His200, Trp102 and Glu103 were suggested to be important for binding of mRNA cap structure, as judged from comparison of the binding abilities of respective mutants with a m7GTP affinity column. Since the crystals of recombinant eIF-4E-m7GTP complex suitable for X-ray crystallography are now in preparation, the detailed interaction mode between them will be opened in near future.

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

Evidence that the 5' end of rabbit globin mRNA is hybridized with its 3' end

1. 1. A radiopolyadenylated rabbit globin mRNA was treated with different concentrations of ribonuclease V 1 from cobra venom. 2. 2. The enzymatic digests were chromatographed on an aminophenylboronate-agarose column, which specifically captured the cap structure i.e. n 7G(5') ppp (5') NmP. 3. 3. When the capture fragment was chromatographed on a Sephadex G-100 column, its size was smaller than the native molecule and also bore radioactivity, i.e. a poly(A) tail. 4. 4. These results provide evidence that the 5' end (which encompasses the cap structure) of rabbit globin mRNA is hybridized and in close proximity to its 3' end. 5. 5. We conclude that this conformation is required for messenger translation efficiency.