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ABSTRACT: Many eukaryotes encode multiple isoforms of the cap-binding translation initiation factor (eIF4E). Leishmania and trypanosomatids encode four paralogs of this protein, but none can complement the eIF4E function in a yeast mutant. A low conservation is observed between the four paralogs, suggesting they assist these organisms survive a multitude of conditions encountered throughout the life cycle. Earlier attempts to decipher their function led to identification of LeishIF4E-4 as the traditional translation initiation factor. LeishIF4E-1 appears to function during thermal stress, via a mechanism not yet understood. LeishIF4E-3 hardly binds cap-4 and thus cannot serve as a typical initiation factor. Although it interacts with an eIF4G homolog, LeishIF4G-4, the two polypeptides do not co-migrate on sucrose gradients. While LeishIF4E-3 enters large particles that increase in size during nutritional stress, LeishIF4G-4 is found only in the top fractions, lighter than the 43S pre-initiation complex. Confocal microscopy localized LeishIF4E-3 (but not LeishIF4G-4) within nutritional stress-induced granules. Accordingly, interaction between the two proteins reduced upon starvation. We therefore propose that under normal conditions, LeishIF4G-4 sequesters LeishIF4E-3 in the cytoplasm. During a nutritional stress, LeishIF4E-3 is modified and released from LeishIF4G-4 to enter stress granules, where inactive mRNAs are stored. Binding of LeishIF4G-4 to LeishIF4E-3 requires a short peptide within the LeishIF4G-4 N-terminus, which bears no similarity to the consensus 4E-binding peptide, YXXXXLΦ. Mutational analysis combined with structure prediction indicates that this interaction is based on an obligatory, conserved α helix in LeishIF4G-4. These features further highlight the uniqueness of LeishIF4E-3 and how it interacts with its binding partners.
RNA biology 11/2012; 9(12). · 5.56 Impact Factor
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ABSTRACT: DED1/VAS belong to the DEAD-box family of RNA helicases that are associated with translation initiation in higher eukaryotes. Here we report on two DED1/VAS homologs that were identified in the genome of Leishmania. The two paralogs include all the domains that are typical of DEAD-box proteins and a phylogenetic analysis suggests that their duplication predates the branching of DED1 and VAS, which took place along with the appearance of early metazoans. The two Leishmania DED1 paralogs complement a yeast strain that fails to express the endogenous DED1, suggesting that they are responsible for a similar function. This is also supported by RNAi-mediated silencing experiments performed in Trypanosoma brucei. The two proteins are functionally redundant, since defects in protein synthesis and cell growth arrest were observed only when both paralogs were eliminated. A partial stage-specific specialization is observed, as LeishDED1-2 is more abundant in promastigotes, whereas expression of LeishDED1-1 increases in amastigotes. Duplication of an essential gene usually offers a safety net against mutations but in this case it also generated two proteins with stage specific expression.
Molecular and Biochemical Parasitology 08/2012; 185(2):127-36. · 2.55 Impact Factor
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ABSTRACT: Trypanosomatids are ancient eukaryotic parasites that migrate between insect vectors and mammalian hosts, causing a range of diseases in humans and domestic animals. Trypanosomatids feature a multitude of unusual molecular features, including polycistronic transcription and subsequent processing by trans-splicing and polyadenylation. Regulation of protein coding genes is posttranscriptional and thus, translation regulation is fundamental for activating the developmental program of gene expression. The spliced-leader RNA is attached to all mRNAs. It contains an unusual hypermethylated cap-4 structure in its 5' end. The cap-binding complex, eIF4F, has gone through evolutionary changes in accordance with the requirement to bind cap-4. The eIF4F components in trypanosomatids are highly diverged from their orthologs in higher eukaryotes, and their potential functions are discussed. The cap-binding activity in all eukaryotes is a target for regulation and plays a similar role in trypanosomatids. Recent studies revealed a novel eIF4E-interacting protein, involved in directing stage-specific and stress-induced translation pathways. Translation regulation during stress also follows unusual regulatory cues, as the increased translation of Hsp83 following heat stress is driven by a defined element in the 3' UTR, unlike higher eukaryotes. Overall, the environmental switches experienced by trypanosomatids during their life cycle seem to affect their translational machinery in unique ways.
Comparative and Functional Genomics 01/2012; 2012:813718. · 1.28 Impact Factor
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ABSTRACT: In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways.
Nucleic Acids Research 07/2011; 39(19):8404-15. · 8.03 Impact Factor
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ABSTRACT: GP63 is an abundant GPI-anchored surface metalloprotease of Leishmania. Jaramillo et al. (2011) show that GP63 manipulates the translation system of host macrophages by cleaving mTOR, which leads to 4E-BP1 dephosphorylation. This study pioneers the observation that Leishmania parasites metabolically paralyze their host cells using an elegant translation shutoff mechanism.
Cell host & microbe 04/2011; 9(4):257-9. · 13.02 Impact Factor
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ABSTRACT: Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3' untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3' UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3' UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5' end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5' UTR, since a hairpin structure abolishes expression of a fused reporter gene.
RNA 02/2010; 16(2):364-74. · 5.09 Impact Factor
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ABSTRACT: Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.
Nucleic Acids Research 04/2009; 37(10):3243-53. · 8.03 Impact Factor
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ABSTRACT: Three-way junctions in folded RNAs have been investigated both experimentally and computationally. The interest in their analysis stems from the fact that they have significantly been found to possess a functional role. In recent work, three-way junctions have been categorized into families depending on the relative lengths of the segments linking the three helices. Here, based on ideas originating from computational geometry, an algorithm is proposed for detecting three-way junctions in data sets of genes that are related to a metabolic pathway of interest. In its current implementation, the algorithm relies on a moving window that performs energy minimization folding predictions, and is demonstrated on a set of genes that are involved in purine metabolism in plants. The pattern matching algorithm can be extended to other organisms and other metabolic cycles of interest in which three-way junctions have been or will be discovered to play an important role. In the test case presented here with, the computational prediction of a three-way junction in Arabidopsis that was speculated to have an interesting functional role is verified experimentally.
In silico biology 02/2008; 8(2):105-20.
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ABSTRACT: Riboswitches are natural RNA sensors that affect gene control via their capacity to bind small molecules. Their prevalence in higher eukaryotes is unclear. We discovered a post-transcriptional mechanism in plants that uses a riboswitch to control a metabolic feedback loop through differential processing of the precursor RNA 3' terminus. When cellular thiamin pyrophosphate (TPP) levels rise, metabolite sensing by the riboswitch located in TPP biosynthesis genes directs formation of an unstable splicing product, and consequently TPP levels drop. When transformed in plants, engineered TPP riboswitches can act autonomously to modulate gene expression. In an evolutionary perspective, a TPP riboswitch is also present in ancient plant taxa, suggesting that this mechanism is active since vascular plants emerged 400 million years ago.
Genes & Development 12/2007; 21(22):2874-9. · 11.66 Impact Factor
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ABSTRACT: The discovery of natural RNA sensors that respond to a change in the environment by a conformational switch can be utilized for various biotechnological and nanobiotechnological advances. One class of RNA sensors is the riboswitch: an RNA genetic control element that is capable of sensing small molecules, responding to a deviation in ligand concentration with a structural change. Riboswitches are modularly built from smaller components. Computational methods can potentially be utilized in assembling these building block components and offering improvements in the biochemical design process. We describe a computational procedure to design RNA switches from building blocks with favorable properties. To achieve maximal throughput for genetic control purposes, future designer RNA switches can be assembled based on a computerized preprocessing buildup of the constituent domains, namely the aptamer and the expression platform in the case of a synthetic riboswitch. Conformational switching is enabled by the RNA versatility to possess two highly stable states that are energetically close to each other but topologically distinct, separated by an energy barrier between them. Initially, computer simulations can produce a list of short sequences that switch between two conformers when trigerred by point mutations or temperature. The short sequences should possess an additional desirable property; when these selected small RNA switch segments are attached to various aptamers, the ligand binding mechanism should replace the aforementioned event triggers, which will no longer be effective for crossing the energy barrier. In the assembled RNA sequence, energy minimization folding predictions should then show no difference between the folded structure of the entire sequence relative to the folded structure of each of its constituents. Moreover, energy minimization methods applied on the entire sequence could aid at this preprocessing stage by exhibiting high mutational robustness to capture the stability of the formed hairpin in the expression platform. The above computer-assisted assembly procedure together with application specific considerations may further be tailored for therapeutic gene regulation. Index Terms-Design of RNA switches, energy minimization methods, RNA folding predictions.
IEEE Transactions on NanoBioscience 03/2007; 6(1):4-11. · 1.28 Impact Factor
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ABSTRACT: The unique structure of 5' mRNA cap from Trypanosomatids is the most modified cap found in nature. Here we present the synthesis of cap-4 (m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up) on a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then previously described. Moreover such modified resin could be a useful tool for affinity purification of Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of phosphorylated tetranucleotide with activated 7-methylguanosine 5'-diphosphate two systems were compared. Surprisingly, the coupling in water with Mn(2+) as a catalyst, gave better results than usually more effective coupling in DMF with ZnCl(2).
Nucleosides Nucleotides & Nucleic Acids 02/2007; 26(10-12):1329-33. · 0.90 Impact Factor
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ABSTRACT: Synthesis of Leishmania mRNA 5'-cap analogs, m(7)Gpppm(2)(6)AmpAm (cap-2), and m(7)Gpppm(2)(6)AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).
Nucleosides Nucleotides & Nucleic Acids 02/2007; 26(10-12):1339-48. · 0.90 Impact Factor
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ABSTRACT: The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.
Eukaryotic Cell 01/2007; 5(12):1969-79. · 3.60 Impact Factor
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ABSTRACT: We previously proposed a mechanism for control of Rubisco expression and assembly during oxidative stress in Chlamydomonas reinhardtii. The N terminus of the large subunit (LSU) comprises an RNA recognition motif (RRM) that is normally buried in the protein, but becomes exposed under oxidizing conditions when the glutathione pool shifts toward its oxidized form. Thus, de novo translation and assembly of Rubisco LSU stop with similar kinetics and the unpaired small subunit (SSU) is rapidly degraded. Here we show that the structure of the N-terminal domain is highly conserved throughout evolution, despite its relatively low sequence similarity. Furthermore, Rubisco from a broad evolutionary range of photosynthetic organisms binds RNA under oxidizing conditions, with dissociation constant values in the nanomolar range. In line with these observations, oxidative stress indeed causes a translational arrest in land plants as well as in Rhodospirillum rubrum, a purple bacterium that lacks the SSU. We highlight an evolutionary conserved element located within alpha-helix B, which is located in the center of the RRM and is also involved in the intramolecular interactions between two LSU chains. Thus, assembly masks the N terminus of the LSU hiding the RRM. When assembly is interrupted due to structural changes that occur under oxidizing conditions or in the absence of a dedicated chaperone, the N-terminal domain can become exposed, leading to the translational arrest of Rubisco LSU. Taken together, these results support a model by which LSU translation is governed by its dimerization. In the case that regulation of type I and type II Rubisco is conserved, the SSU does not appear to be directly involved in LSU translation.
Plant physiology 08/2006; 141(3):1089-97. · 6.53 Impact Factor
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ABSTRACT: Oxidative stress in plants and green algae has multiple damaging effects, and leads to the degradation of Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco). We recently showed for the green algae Chlamydomonas reinhardtii that in response to a photo-oxidative stress, nascent synthesis of its chloroplast encoded large subunit (LSU) stops. In parallel, newly synthesized small subunits (SSU) that are encoded by the nucleus are rapidly degraded, thus assembly of new holoenzyme particles is inhibited. Here we show that under extreme oxidizing conditions, the steady-state level of the SSU is also reduced. Cleavage of the LSU under oxidizing conditions is well established, and we show, using sucrose gradients, that the resulting fragments of the LSU co-exist as parts of the holoenzyme. In parallel, we demonstrate the selective in-vivo formation of high-density aggregates of Rubisco particles, in response to oxidative stress. Given the known tendency of unassembled LSUs to aggregate, we propose that the rapid elimination of the SSU during oxidative stress along with the fragmentation of the LSU and formation of intra-protein disulfide bridges, leads to the observed aggregation of Rubisco particles. Indeed, we note here a substantially decreased ratio of SSU in the aggregated Rubisco particles. We also observed that this aggregation marks the viability threshold of C. reinhardtii cells exposed to oxidative stress.
Planta 12/2005; 222(5):787-93. · 3.00 Impact Factor
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ABSTRACT: In Chlamydomonas reinhardtii, a light-induced oxidative stress shifts the glutathione pool toward its oxidized form, resulting in a translational arrest of the large subunit (LSU) of Rubisco. We show here that the translational arrest of LSU is tightly coordinated with cessation of Rubisco assembly, and both processes take place after a threshold level of reactive oxygen species is reached. As a result, the small subunit is also eliminated by rapid degradation. We previously showed that the amino terminus of the LSU could bind RNA in a sequence-independent manner, as it shares a structural similarity with the RNA recognition motif. This domain becomes exposed only under oxidizing conditions, thus restricting the RNA-binding activity. Here we show that in vitro, thiol groups of both subunits become oxidized in the presence of oxidized glutathione. The structural changes are mediated by oxidized glutathione, whereas only very high concentrations of H2O2 confer similar results in vitro. Changes in the redox state of the LSU thiol groups are also observed in vivo, in response to a physiological light shock caused by transfer of cells from low light to high light. We propose that during a photooxidative stress, oxidation of thiol groups occurs already in nascent LSU chains, perhaps hindering their association with chaperones. As a result, their RNA recognition motif domain becomes exposed and will bind any RNA in its vicinity, including its own transcript. Due to this binding the ribosome stalls, preventing the assembly of additional ribosomes on the transcript. Polysome analysis using Suc gradients indeed shows that the rbcL RNA is associated with the polysomal fraction at all times but shifts toward fractions that contain smaller polysomes and monosomes during oxidative stress. Thus, translational arrest of the LSU most likely occurs at a postinitiation stage.
Plant physiology 03/2005; 137(2):738-46. · 6.53 Impact Factor
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ABSTRACT: We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.
Applied and Environmental Microbiology 01/2005; 70(12):7156-60. · 3.83 Impact Factor
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Fourth International IEEE Computer Society Computational Systems Bioinformatics Conference Workshops & Poster Abstracts (CSB 2005 Workshops), 8-11 August 2005, Stanford, CA, USA; 01/2005
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ABSTRACT: All eukaryotic mRNAs possess a 5'-cap (m(7)GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5'-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher K(as) for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m(7)GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both ligands. A sequence alignment shows that LeishIF4E-1 lacks the region that parallels the C terminus in the murine eIF4E. Truncation of this region in the mouse protein reduces the difference that is observed between its binding to m(7)GTP and cap-4, prior to this deletion. We hypothesize that variations in the structure of LeishIF4E-1, possibly also the absence of a region that is homologous to the C terminus of the mouse protein, promote its ability to interact with the cap-4 structure. LeishIF4E-1 is distributed in the cytoplasm, but its function is not clear yet, because it cannot substitute the mammalian eIF4E in a rabbit reticulocyte in vitro translation system.
RNA 12/2004; 10(11):1764-75. · 5.09 Impact Factor
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ABSTRACT: Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5' end of all mRNAs, thus donating a 5' cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5'-5'-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5' cap includes 2'-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7Gpppm3(6,6,2')Apm2'Apm2' Cpm2(3,2')U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5' terminal mRNA tetranucleotide fragment (pm3(6,6,2')Apm2'Apm2'Cpm2(3,2')U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5'-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.
RNA 10/2004; 10(9):1469-78. · 5.09 Impact Factor
