Translation and Translational Control in Dinoflagellates.

Dinoflagellates are unicellular protists that feature a multitude of unusual nuclear features, including large genomes, packaging of DNA without histones, and multiple gene copies organized as tandem gene arrays. Furthermore, all dinoflagellate mRNAs experience trans-splicing with a common 22-nucleotide splice leader (SL) sequence. These features challenge some of the concepts and assumptions about the regulation of gene expression derived from work on model eukaryotes such as yeasts and mammals. Translational control in the dinoflagellates, based on extensive study of circadian bioluminescence and by more recent microarray and transcriptome analyses, is now understood to be a crucial element in regulating gene expression. A picture of the translation machinery of dinoflagellates is emerging from the recent availability of transcriptomes of multiple dinoflagellate species and the first complete genome sequences. The components comprising the translational control toolkit of dinoflagellates are beginning to take shape and are outlined here.

Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation.

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 µg/mL), carbenicillin (100 µg/mL), and streptomycin sulfate (50 µg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments.

Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium.

Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.

The alveolate translation initiation factor 4E family reveals a custom toolkit for translational control in core dinoflagellates.

BACKGROUND: Dinoflagellates are eukaryotes with unusual cell biology and appear to rely on translational rather than transcriptional control of gene expression. The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in regulating gene expression because eIF4E binding to the mRNA cap is a control point for translation. eIF4E is part of an extended, eukaryote-specific family with different members having specific functions, based on studies of model organisms. Dinoflagellate eIF4E diversity could provide a mechanism for dinoflagellates to regulate gene expression in a post-transcriptional manner. Accordingly, eIF4E family members from eleven core dinoflagellate transcriptomes were surveyed to determine the diversity and phylogeny of the eIF4E family in dinoflagellates and related lineages including apicomplexans, ciliates and heterokonts. RESULTS: The survey uncovered eight to fifteen (on average eleven) different eIF4E family members in each core dinoflagellate species. The eIF4E family members from heterokonts and dinoflagellates segregated into three clades, suggesting at least three eIF4E cognates were present in their common ancestor. However, these three clades are distinct from the three previously described eIF4E classes, reflecting diverse approaches to a central eukaryotic function. Heterokonts contain four clades, ciliates two and apicomplexans only a single recognizable eIF4E clade. In the core dinoflagellates, the three clades were further divided into nine sub-clades based on the phylogenetic analysis and species representation. Six of the sub-clades included at least one member from all eleven core dinoflagellate species, suggesting duplication in their shared ancestor. Conservation within sub-clades varied, suggesting different selection pressures. CONCLUSIONS: Phylogenetic analysis of eIF4E in core dinoflagellates revealed complex layering of duplication and conservation when compared to other eukaryotes. Our results suggest that the diverse eIF4E family in core dinoflagellates may provide a toolkit to enable selective translation as a strategy for controlling gene expression in these enigmatic eukaryotes.

Translating the message: Karlodinium veneficum possesses an expanded toolkit of protein translation initiation factors.

Dinoflagellates are unusual eukaryotes with large genomes and a reduced role for transcriptional regulation compared to other eukaryotes. The mRNA in dinoflagellates is trans-spliced with a 5'-spliced-leader sequence, yielding a 22-nucleotide 5'-sequence with a methylated nucleotide cap. Since the control of gene expression is primarily post-transcriptional, this study focuses on mRNA recruitment as a means for regulating gene expression and specifically on the diversity of eIF4E family members. Three novel clades related to the cap binding initiation factor eIF4E have been recognized in alveolates that are distinct from the three metazoan classes of eIF4E. We have analyzed the characteristics of five of the fourteen eIF4E family members from Karlodinium veneficum, four from clade 1 and one from clade 2. Members of each clade all bear the distinctive features of a cap-binding protein. We examined their ability to interact with the cap analogue, m7GTP using an in vitro bead-binding assay. We show that recombinant eIF4E-1 family members are able to bind the cap analogue m7GTP, but eIF4E-2b binds poorly. Overall, the eIF4E-1 family members may be serving as general cap-binding translation initiation factors, while the eIF4E-2 (and perhaps eIF4E-3) family members may serve a regulatory role in gene expression.

Variation in spatial and temporal incidence of the crustacean pathogen Hematodinium perezi in environmental samples from Atlantic Coastal Bays.

BACKGROUND: Hematodinium perezi, a parasitic dinoflagellate, infects and kills blue crabs, Callinectes sapidus, along the Atlantic and Gulf coasts of the United States. The parasite proliferates within host hemolymph and tissues, and also produces free-swimming biflagellated dinospores that emerge from infected crabs. Infections in C. sapidus recur annually, and it is not known if biotic or environmental reservoirs contribute to reinfection and outbreaks. To address this data gap, a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed to asses the temporal and spatial incidence of the parasite in Delaware and Maryland coastal bays. RESULTS: A previously-used PCR assay for H. perezi, based on the small subunit rRNA gene sequence, was found to lack adequate species specificity to discriminate non-Hematodinium sp. dinoflagellate species in environmental samples. A new ITS2-targeted assay was developed and validated to detect H. perezi DNA in sediment and water samples using E. coli carrying the H. perezi rDNA genes. Application of the method to environmental samples identified potential hotspots in sediment in Indian River Inlet, DE and Chincoteague Bay, MD and VA. H. perezi DNA was not detected in co-occurring shrimp or snails, even during an outbreak of the parasite in C. sapidus. CONCLUSIONS: H. perezi is present in water and sediment samples in Maryland and Delaware coastal bays from April through November with a wide spatial and temporal variability in incidence. Sampling sites with high levels of H. perezi DNA in both bays share characteristics of silty, organic sediments and low tidal currents. The environmental detection of H. perezi in spring, ahead of peak prevalence in crabs, points to gaps in our understanding of the parasite's life history prior to infection in crabs as well as the mode of environmental transmission. To better understand the H. perezi life cycle will require further monitoring of the parasite in habitats as well as hosts. Improved understanding of potential environmental transmission to crabs will facilitate the development of disease forecasting.

Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists.

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus.

Mortality among blue crab Callinectes sapidus in soft shell production facilities is typically 25% or greater. The harvest, handling, and husbandry practices of soft shell crab production have the potential to spread or exacerbate infectious crab diseases. To investigate the possible role of viruses in soft shell crab mortalities, we took advantage of the physicochemical properties of double-stranded RNA (dsRNA) to isolate a putative virus genome. Further characterization confirmed the presence of a reo-like virus that possesses 12 dsRNA genome segments. The virus was present in >50% of dead or dying soft shell crabs, but fewer than 5% of healthy hard crabs. Injection of the virus caused mortality and resulted in the appearance of viral RNA and virus inclusions in hemocytes. The genome of the virus was partially sequenced and the information used to develop a reverse transcription polymerase chain reaction (RT-PCR) assay that is able to detect the virus genome in as little as 7.5 pg of total RNA. The molecular tools developed during this study will allow us to quantify prevalence of the blue crab reo-like virus in captive (soft shell facilities, aquaculture operations) and wild populations and facilitate understanding of the role this virus has in blue crab life history.

The Atlantic salmon Z-DNA binding protein kinase phosphorylates translation initiation factor 2 alpha and constitutes a unique orthologue to the mammalian dsRNA-activated protein kinase R.

The translation initiation factor 2 alpha (eIF2alpha)-kinase, dsRNA-activated protein kinase (PKR), constitutes one of the major antiviral proteins activated by viral infection of vertebrates. PKR is activated by viral double-stranded RNA and subsequently phosphorylates the alpha-subunit of translation initiation factor eIF2. This results in overall down regulation of protein synthesis in the cell and inhibition of viral replication. Fish appear to have a PKR-like protein that has Z-DNA binding domains instead of dsRNA binding domains in the regulatory domain, and has thus been termed Z-DNA binding protein kinase (PKZ). We present the cloning of the Atlantic salmon PKZ cDNA and show its upregulation by interferon in Atlantic salmon TO cells and poly inosinic poly cytodylic acid in head kidney. We also demonstrate that recombinant Atlantic salmon PKZ, expressed in Escherichia coli, phosphorylates eIF2alphain vitro. This is the first demonstration that PKZ is able to phosphorylate eIF2alpha. PKZ activity, as measured by phosphorylation of eIF2alpha, was increased after addition of Z-DNA, but not by dsRNA. In addition, we show that wild-type Atlantic salmon PKZ, but not the kinase defective variant K217R, has a direct inhibitory effect on protein synthesis after transient expression in Chinook salmon embryo cells. Overall, the results support a role for PKZ, like PKR, in host defense against virus infection.

Approaches for analyzing the differential activities and functions of eIF4E family members.

The translational initiation factor eIF4E binds to the m(7)G-containing cap of mRNA and participates in recruitment of mRNA to ribosomes for protein synthesis. eIF4E also functions in nucleocytoplasmic transport of mRNA, sequestration of mRNA in a nontranslatable state, and stabilization of mRNA against decay in the cytosol. Multiple eIF4E family members have been identified in a wide range of organisms that includes plants, flies, mammals, frogs, birds, nematodes, fish, and various protists. This chapter reviews methods that have been applied to learn the biochemical properties and physiological functions that differentiate eIF4E family members within a given organism. Much has been learned to date about approaches to discover new eIF4E family members, their in vitro properties (cap binding, stimulation of cell-free translation systems), tissue and developmental expression patterns, protein-binding partners, and their effects on the translation or repression of specific subsets of mRNA. Despite these advances, new eIF4E family members continue to be found and new physiological roles discovered.

Phylogenetic analysis of eIF4E-family members.

BACKGROUND: Translation initiation in eukaryotes involves the recruitment of mRNA to the ribosome which is controlled by the translation factor eIF4E. eIF4E binds to the 5'-m7Gppp cap-structure of mRNA. Three dimensional structures of eIF4Es bound to cap-analogues resemble 'cupped-hands' in which the cap-structure is sandwiched between two conserved Trp residues (Trp-56 and Trp-102 of H. sapiens eIF4E). A third conserved Trp residue (Trp-166 of H. sapiens eIF4E) recognizes the 7-methyl moiety of the cap-structure. Assessment of GenBank NR and dbEST databases reveals that many organisms encode a number of proteins with homology to eIF4E. Little is understood about the relationships of these structurally related proteins to each other. RESULTS: By combining sequence data deposited in the Genbank databases, we have identified sequences encoding 411 eIF4E-family members from 230 species. These sequences have been deposited into an internet-accessible database designed for sequence comparisons of eIF4E-family members. Most members can be grouped into one of three classes. Class I members carry Trp residues equivalent to Trp-43 and Trp-56 of H. sapiens eIF4E and appear to be present in all eukaryotes. Class II members, possess Trp-->Tyr/Phe/Leu and Trp-->Tyr/Phe substitutions relative to Trp-43 and Trp-56 of H. sapiens eIF4E, and can be identified in Metazoa, Viridiplantae, and Fungi. Class III members possess a Trp residue equivalent to Trp-43 of H. sapiens eIF4E but carry a Trp-->Cys/Tyr substitution relative to Trp-56 of H. sapiens eIF4E, and can be identified in Coelomata and Cnidaria. Some eIF4E-family members from Protista show extension or compaction relative to prototypical eIF4E-family members. CONCLUSION: The expansion of sequenced cDNAs and genomic DNAs from all eukaryotic kingdoms has revealed a variety of proteins related in structure to eIF4E. Evolutionarily it seems that a single early eIF4E gene has undergone multiple gene duplications generating multiple structural classes, such that it is no longer possible to predict function from the primary amino acid sequence of an eIF4E-family member. The variety of eIF4E-family members provides a source of alternatives on the eIF4E structural theme that will benefit structure/function analyses and therapeutic drug design.

Characterization of mammalian eIF4E-family members.

The translational factor eukaryotic initiation factor 4E (eIF4E) is a central component in the initiation and regulation of translation in eukaryotic cells. Through its interaction with the 5' cap structure of mRNA, eIF4E functions to recruit mRNAs to the ribosome. The accumulation of expressed sequence tag sequences has allowed the identification of three different eIF4E-family members in mammals termed eIF4E-1, eIF4E-2 (4EHP, 4E-LP) and eIF4E-3, which differ in their structural signatures, functional characteristics and expression patterns. Unlike eIF4E-1, which is found in all eukaryotes, orthologues for eIF4E-2 appear to be restricted to metazoans, while those for eIF4E-3 have been found only in chordates. Like prototypical eIF4E-1, eIF4E-2 was found to be ubiquitously expressed, with the highest levels in the testis. Expression of eIF4E-3 was detected only in heart, skeletal muscle, lung and spleen. Similarly to eIF4E-1, both eIF4E-2 and eIF4E-3 can bind to the mRNA cap-structure. However, in contrast to eIF4E-1 which interacts with both the scaffold protein, eIF4G and the translational repressor proteins, the eIF4E-binding proteins (4E-BPs), eIF4E-2 and eIF4E-3 each possesses a range of partial activities. eIF4E-2 does not interact with eIF4G, but does interact with 4E-BPs. Conversely, eIF4E-3 interacts with eIF4G, but not with 4E-BPs. Neither eIF4E-2 nor eIF4E-3 is able to rescue the lethality of eIF4E gene deletion in yeast. It is hypothesized that each eIF4E-family member fills a specialized niche in the recruitment of mRNAs by the ribosome through differences in their abilities to bind cap and/or to interact with eIF4G and the 4E-BPs.

Two zebrafish eIF4E family members are differentially expressed and functionally divergent.

Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.

Characterization of rainbow trout and zebrafish eukaryotic initiation factor 2alpha and its response to endoplasmic reticulum stress and IPNV infection.

The cDNAs of rainbow trout and zebrafish eIF2alpha have been isolated and found to encode proteins of similar molecular weight and isoelectric point to the alpha-subunit of the human translational initiation factor, eIF2. The rainbow trout (36.0kDa) and zebrafish (36.2kDa) eIF2alphas share 93 and 91% identity to the human protein, respectively, and are recognized by antibodies raised to the human form. In mammals, the phosphorylation of the alpha-subunit of eIF2 plays a key role in the regulation of protein synthesis in response to a range of cellular stresses. Regions corresponding to the human phosphorylation and kinase-docking sites are identical in the proteins of both fish species, as are residues that interact with the eIF2 recycling factor, eIF2B. Moreover, both recombinant rainbow trout and zebrafish eIF2alphas can be phosphorylated in vitro by the mammalian heme-sensitive eIF2alpha-kinase, HRI/HCR, as well as the interferon-inducible, dsRNA sensitive kinase, PKR. Phosphorylation of rainbow trout and zebrafish eIF2alpha can also occur in vivo. RTG-2 and ZFL cells subjected to endoplasmic reticulum (ER) stress by treatment with the Ca(2+)-ionophore A23187 showed increased levels of eIF2alpha phosphorylation, suggesting similarity between the ER stress response in fish and other higher eukaryotes. Furthermore, RTG-2 cells responded to treatment with poly(I).poly(C) or to infection by infectious pancreatic necrosis virus, IPNV, by increasing eIF2alpha phosphorylation. These data imply that RTG-2 cells express the interferon-induced eIF2alpha-kinase, PKR and suggests that the interferon/eIF2alpha/PKR response to virus infection may be a conserved vertebrate characteristic. Overall these data are consistent with the premise that fish are able to regulate protein synthesis in response to cellular stresses through phosphorylation of eIF2alpha.

Overview of eukaryotic in vitro translation and expression systems.

The ability to investigate cellular processes in vitro permits detailed analysis of the process and its molecular components. Eukaryotic translation and expression is one system that has been well studied. This overview describes the development of in vitro systems, including such approaches as continuous-flow systems, coupled transcription/translation, and the incorporation of non-natural amino acids. It also discusses molecular and genetic studies to probe translation, including post-translational fate of the synthesized proteins.

Yeast "knockout-and-rescue" system for identification of eIF4E-family members possessing eIF4E-activity.

Evidence from several laboratories and sequencing projects has revealed that many eukaryotes contain multiple proteins related in sequence to the human mRNA-cap binding translation initiation factor 4E (eIF4E-1). Although some have been shown to bind cap-analogues, whether all eIF4E-family members function as translation initiation factors is unclear Furthermore, the existence of proteins related to eIF4E complicates the identification of the translation factor by sequence-based approaches. Methods to assess the functionality of eIF4E are limited. The most informative, single assay to identify proteins with eIF4E-activity is that of rescue of the lethal disruption of the single Saccharomyces cerevisiae eIF4E gene. We have developed a simplified yeast eIF4E "knockout-and-rescue" system, the characteristics of which are (i) a haploid system that obviates the needfor a "plasmid shuffle", (ii) a simple G418-based selection for yeast lacking a chromosomal eIF4E gene, and (iii) a glucose-based selection to deplete the strain of a human eIF4E-1 substitute and to assess the eIF4E-activity of an untested elF4E-family member In this form, the yeast eIF4E knockout-and-rescue system becomes a tool available to any laboratory experienced in the selection of microbial strains with antibiotics and standard media for the identification and isolation of cDNAs encoding proteins with eIF4E-activity.

Phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation.

The long uORF-burdened 5'UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet-derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES-mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c-Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES-mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2 alpha. Furthermore, IRES use is decreased by over-expression of the dominant-negative form of the eIF2 alpha kinase, PKR, the vaccinia virus K3L gene, or the eIF2 alpha-S51A variant which result in decreased eIF2 alpha phosphorylation. These data demonstrate a connection between eIF2 alpha phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2 alpha, known to be important for cap-dependent translational control, serves to fine-tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES-containing mRNAs under a range of physiological circumstances.